BackgroundAdjuvant immune checkpoint inhibitors (ICI) have improved survival in stage III cutaneous melanoma, yet many patients do not benefit. The tumor microenvironment is pivotal for durable responses; defining cellular composition can pinpoint immune components promoting anti-tumor activity and identify biomarkers associated with improved outcomes.MethodRegional lymph nodes (RLNs) were obtained at surgery from 29 patients with stage III melanoma. Eligible patients received adjuvant anti-PD-1 (αPD-1; pembrolizumab or nivolumab). Mass cytometry (CyTOF) was used to determine cellular composition of pre-treatment surgical specimens. NanoString bulk gene expression data from 125 patients receiving surgery without adjuvant therapy were used to evaluate trends observed in the CyTOF dataset.ResultsHigher proportions of CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cells were associated with improved prognosis in the CyTOF cohort. In the untreated cohort, a “B cell excluded” subgroup (< 2.5% tumor-infiltrating lymphocyte pathology score) had worse outcome, showing reduced B cell score and lower expression of activation genes including CD38, without change in CD8+ T cell score.ConclusionBaseline infiltration of CD8+ TRM and plasmablast-like CD38hiCD19dim cells in RLN is strongly associated with prolonged distant metastasis-free survival in patients receiving αPD-1, supporting their potential as prognostic biomarkers in stage III melanoma.

Adjuvant immunotherapy has significantly improved survival for patients with stage III cutaneous melanoma, yet a fraction of patients will not benefit from immune checkpoint inhibitors (ICI). The tumor microenvironment plays a pivotal role in generating durable responses to ICI. By analyzing the cellular composition of tumor-associated subsets, key immune components essential for promoting an anti-tumor environment can be pinpointed. This will allow for both patient stratification and identification of biomarkers associated with improved patient outcome. Regional lymph nodes were obtained from patients with stage III melanoma at surgery (n=29). Patients eligible for anti-PD-1 therapy (αPD-1; pembrolizumab or nivolumab) received adjuvant treatment for up to one year. CyTOF was used to determine cellular composition in pre-treatment surgical specimens. Bulk gene expression data generated by NanoString from patients receiving surgery without adjuvant therapy (n=125) was implemented for evaluating trends observed in the CyTOF dataset. Although no significant differences were observed across major hierarchical immune cell types between patients who developed distant metastasis after surgery and those that did not, an increased proportion of CD103 + PD-1 + CD8 + (T RM ) T cells and plasmablast-like CD38 hi CD19 dim cells were associated with improved prognosis in the CyTOF cohort. In the untreated cohort, a subset of patients defined as “Ultra-cold” (< 2.5 % tumor-infiltrating lymphocyte (TIL) scored by a pathologist) had significantly worse outcome than those with higher TIL infiltration. This Ultra-cold TIL group was associated with reduced B cell score, but not CD8 + T cell score, as well as reduced expression of activation genes like CD38 . In this study, CD103 + PD-1 + CD8 + (T RM ) T cells and plasmablast-like CD38 hi CD19 dim cell populations were found to be strongly associated with prolonged distant metastasis-free survival in regional lymph nodes from patients with stage III melanoma treated with αPD-1. This suggests an association between progression and infiltration of these cell types at baseline and highlights the potential of using immune cell subsets as prognostic biomarkers. What is already known on this topic – Patients being diagnosed with stage III melanoma will receive immune checkpoint inhibitors but will often be cured by surgery alone. Selection of which patients might benefit from treatment is still unresolved. Accurate biomarkers would aid in treatment stratification, to avoid overtreatment, and unnecessary toxicities. What this study adds – This study highlights how baseline CD103 + PD-1 + CD8 + (T RM ) T cells and plasmablast-like CD38 hi CD19 dim cell populations in the regional lymph node, is strongly associated with improved outcome in patients receiving anti-PD-1 therapy. How this study might affect research, practice or policy – The strong association between baseline plasmablast-like cell infiltration in RLN and prolonged distant metastasis free- survival, highlights this cell type as a potential treatment stratification criterion to identify patients with good prognosis.

Background:The tumor microenvironment influences treatment response in ER-positive breast cancer, but what distinguishes responders from non-responders and how it changes during treatment is poorly understood. Methods:ER-positive breast tumors treated with neoadjuvant chemotherapy with or without bevacizumab were profiled with bulk proteomics pre- (n = 95), on- (n = 84) and post-treatment (n = 100). A subset of tumors was profiled with spatial single-cell proteomics pre- (n = 13) and on-treatment (n = 11). Cell phenotypes, spatial location and activation states were determined, and cellular colocalization assessed with spatial metrics. Bulk and spatial features were evaluated against treatment response defined by residual cancer burden. Results:Treatment with bevacizumab amplified chemotherapy effects on proteomic signaling. The immune contexture shifted from suppressive to supportive during treatment through decreased macrophage, regulatory and anergic T-cell density and increased colocalization between epithelial cells and CD8+, CD4+ T-cells and dendritic cells. At baseline, responders had high density of effector memory T-cells, while non-responders had more naïve T-cells. In addition, responders had increased colocalization of epithelial cells with macrophages, and effector memory T-cells with M1-like macrophages compared to non-responders. Conclusions:Spatially distinct tumor-immune microenvironments influence response to neoadjuvant treatment, offering valuable insights for guiding treatment decisions.

Background Adjuvant immunotherapy has significantly improved survival for patients with stage III cutaneous melanoma, yet a fraction of patients will not benefit from immune checkpoint inhibitors (ICI). The tumor microenvironment plays a pivotal role in generating durable responses to ICI. By analyzing the cellular composition of tumor-associated subsets, key immune components essential for promoting an anti-tumor environment can be pinpointed. This will allow for both patient stratification and identification of biomarkers associated with improved patient outcome. Method Regional lymph nodes were obtained from patients with stage III melanoma at surgery (n=29). Patients eligible for anti-PD-1 therapy (αPD-1; pembrolizumab or nivolumab) received adjuvant treatment for up to one year. CyTOF was used to determine cellular composition in pre-treatment surgical specimens. Bulk gene expression data generated by NanoString from patients receiving surgery without adjuvant therapy (n=125) was implemented for evaluating trends observed in the CyTOF dataset. Results Although no significant differences were observed across major hierarchical immune cell types between patients who developed distant metastasis after surgery and those that did not, an increased proportion of CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cells were associated with improved prognosis in the CyTOF cohort. In the untreated cohort, a subset of patients defined as “Ultra-cold” (< 2.5 % tumor-infiltrating lymphocyte (TIL) scored by a pathologist) had significantly worse outcome than those with higher TIL infiltration. This Ultra-cold TIL group was associated with reduced B cell score, but not CD8+ T cell score, as well as reduced expression of activation genes like CD38 . Conclusion In this study, CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cell populations were found to be strongly associated with prolonged distant metastasis-free survival in regional lymph nodes from patients with stage III melanoma treated with αPD-1. This suggests an association between progression and infiltration of these cell types at baseline and highlights the potential of using immune cell subsets as prognostic biomarkers. What is already known on this topic – Patients being diagnosed with stage III melanoma will receive immune checkpoint inhibitors but will often be cured by surgery alone. Selection of which patients might benefit from treatment is still unresolved. Accurate biomarkers would aid in treatment stratification, to avoid overtreatment, and unnecessary toxicities. What this study adds – This study highlights how baseline CD103+PD-1+CD8+ (TRM) T cells and plasmablast-like CD38hiCD19dim cell populations in the regional lymph node, is strongly associated with improved outcome in patients receiving anti-PD-1 therapy. How this study might affect research, practice or policy – The strong association between baseline plasmablast-like cell infiltration in RLN and prolonged distant metastasis free- survival, highlights this cell type as a potential treatment stratification criterion to identify patients with good prognosis. ### Competing Interest Statement The authors have declared no competing interest. Southern and Eastern Norway Regional Health Authority, https://ror.org/02qx2s478, 2019048, 2016117, 2017100 Norwegian Ministry of Health and Care Services Fondsstiftelsen at Oslo University Hospital

Chemotherapy is an integral part of the clinical management of triple negative breast cancer (TNBC), however, development of chemoresistance occurs frequently. Tumor sensitivity to treatment is known to be strongly influenced by the immune microenvironment, signifying the predictive potential of immune alterations. How tumor cells that acquire resistance may subsequently modulate the immune microenvironment it is still not well described. Here, we investigated immunomodulation in the context of acquired chemoresistance in TNBC, focusing on the role of the secretome. Bulk RNA sequencing and multiplex cytokine profiling were performed on paclitaxel-resistant and -sensitive isogenic variants of TNBC cells to reveal resistance-associated secretome alterations. The immunomodulatory influence of the tumor cell secretome was investigated by exploring its effect on monocytes, macrophages (MΦs) and T cells derived from healthy blood donors. The influence on the immune cell phenotype and activity was evaluated by measuring molecular markers and performing functional assays. To validate the clinical relevance, we utilized longitudinal -omics data from breast cancer patients refractory to standard chemotherapy in the NeoAva clinical trial. CIBERSORT was applied to transcriptomics data to infer MΦ and T cell abundance in individual tumors upon treatment. To evaluate their association with the secretome profiles, patient-matched serum cytokine data were used. The acquisition of chemoresistance was accompanied by enhanced secretion of cytokines. Subsequently, the resistant cell secretome affected the abundance, phenotype and activity of immune cells. Specifically, it potentiated the recruitment of monocytes, facilitated the polarization of MΦs towards the immunosuppressive M2-like phenotype, and attenuated the activation of CD8+ T cells. Data from the NeoAva clinical cohort validated the enrichment of M2 MΦs and/or the depletion of M1 MΦs after treatment in the majority of residual tumors. The MΦ-associated changes counteracted CD8+ T cell abundance and were partially associated with the cytokine-enriched secretome. Development of chemoresistance in BC is associated with alterations in the tumor secretome, which impairs immune activation and facilitates immunosuppression. Knowledge on the immune microenvironment in residual tumors after standard chemotherapy could aid in selecting rational treatment options for this group of patients.

Abstract Triple Negative Breast Cancer (TNBC) is a BC subgroup with the least favorable outcome and limited therapeutic options. Chemotherapy has long been the standard treatment, but resistance often develops. In recent years, various immunotherapy approaches have diversified, including combinations of chemo and immunotherapies. Increased understanding on how the difference in chemosensitivity affects the immune cells is of importance for further development of chemo-immunotherapies for TNBC. In this project we investigate how TNBC with distinct chemosensitivity modulate the immune microenvironment. Specifically, we explore the influence of the secretome, derived from the chemoresistant versus the sensitive BC cells, on macrophages (MØs) and T-cells. We have disclosed the chemosensitivity-dependent differences in the secretome, which were associated with diverse effects on MØ recruitment, polarization, and activation. Currently we investigate the secretome influence on interferon (IFN)-activated MØ. IFN stimuli is known to trigger tumoricidal activities in MØ, and we are setting up a co-culture assay, where such MØ functions could be explored in vitro. To study the secretome influence on T-cells, we employ T cell receptor (TCR)-engineered T cells that become activated in the presence of target cells. The preliminary results indicate that activation of the TCR T-cells was reduced in the presence of the “chemoresistant” secretome, suggesting its immunosuppressive influence. Work is in progress to validate this observation using other TCR T cells and functional markers of active T cells. Citation Format: Eleni Skourti, Kotryna Seip, Eivind V. Egeland, Shakila Jabeen, Nadia Mensali, Inger Øynebråten, Siri Juell, Else M. Inderberg, Olav Engebråten, Alexandre B. Corthay, Gunhild M. Mælandsmo, Lina Prasmickaite. Breast cancer with different chemosensitivity has distinct influence on the immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5554.

Background:Biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs) are receiving increasing attention in anti-cancer nanomedicine development not only for targeted cancer chemotherapy, but also for modulation of the tumor microenvironment. We previously reported promising results with cabazitaxel (CBZ) loaded poly(2-ethylbutyl cyanoacrylate) NPs (PEBCA-CBZ NPs) in a patient derived xenograft (PDX) model of triple-negative breast cancer, and this was associated with a decrease in M2 macrophages. The present study aims at comparing two endotoxin-free PACA NP variants (PEBCA and poly(2-ethylhexyl cyanoacrylate); PEHCA), loaded with CBZ and test whether conjugation with folate would improve their effect.Methods:Cytotoxicity assays and cellular uptake of NPs by flow cytometry were performed in different breast cancer cells. Biodistribution and efficacy studies were performed in PDX models of breast cancer. Tumor associated immune cells were analyzed by multiparametric flow cytometry.Results:In vitro studies showed similar NP-induced cytotoxicity patterns despite difference in early NP internalization. On intravenous injection, the liver cleared the majority of NPs. Efficacy studies in the HBCx39 PDX model demonstrated an enhanced effect of drug-loaded PEBCA variants compared with free drug and PEHCA NPs. Furthermore, the folate conjugated PEBCA variant did not show any enhanced effects compared with the unconjugated counterpart which might be due to unfavorable orientation of folate on the NPs. Finally, analyses of the immune cell populations in tumors revealed that treatment with drug loaded PEBCA variants affected the myeloid cells, especially macrophages, contributing to an inflammatory, immune activated tumor microenvironment.Conclusion:We report for the first time, comparative efficacy of PEBCA and PEHCA NP variants in triple negative breast cancer models and show that CBZ-loaded PEBCA NPs exhibit a combined effect on tumor cells and on the tumor associated myeloid compartment, which may boost the anti-tumor response.

BACKGROUND:Resistance to chemotherapy, combined with heterogeneity among resistant tumors, represents a significant challenge in the clinical management of triple negative breast cancer (TNBC). By dissecting molecular pathways associated with treatment resistance, we sought to define patient sub-groups and actionable targets for next-line treatment. METHODS:Bulk RNA sequencing and reverse phase protein array profiling were performed on isogenic patient-derived xenografts (PDX) representing paclitaxel-sensitive and -resistant tumors. Pathways identified as upregulated in the resistant model were further explored as targets in PDX explants. Their clinical relevance was assessed in two distinct patient cohorts (NeoAva and MET500). RESULTS:Increased activity in signaling pathways involving SRC-family kinases (SFKs)- and MAPK/ERK was found in treatment resistant PDX, with targeted inhibitors being significantly more potent in resistant tumors. Up-regulation of SFKs- and MAPK/ERK-pathways was also detected in a sub-group of chemoresistant patients after neoadjuvant treatment. Furthermore, High SFK expression (of either SRC, FYN and/or YES1) was detected in metastatic lesions of TNBC patients with fast progressing disease (median disease-free interval 27 vs 105 months). CONCLUSIONS:Upregulation of SFK-signaling is found in a subset of chemoresistant tumors and is persistent in metastatic lesions. Based on pre-clinical results, these patients may respond favorably to treatment targeting SFKs.

Background and Purpose: Neoadjuvant treatment regimens containing the DNA-targeting chemotherapy carboplatin has been shown to increase the pathological complete response (pCR) rate in triple negative (TN) breast cancer. In addition, the response to such therapy among patients with aggressive hormone receptor positive (HR+) cancer should be investigated. Neither the molecular mechanisms leading to good response, nor the criteria for selection of optimal treatment groups are known. Therefore, predictive biomarkers that identify responders to carboplatin in breast cancer are needed. Materials and Methods: In the I-BCT phase II clinical trial (ClinicalTrials.gov identifier: NCT02546232), HER2-negative breast cancer patients (N = 187) were randomized (1:1) to receive neoadjuvant chemotherapy with or without carboplatin. Pre-treatment tumor biopsies (N = 178, N = 89 in each arm) were analyzed by reverse-phase protein array (RPPA) for expression levels of 494 breast cancer relevant (phospho-) proteins. Change in tumor size during treatment was used as continuous response measure. In addition, response was dichotomized with 30 % tumor shrinkage as cutoff. For the patients that received carboplatin, differential expression analysis between responders and non-responders was performed to identify proteins that may predict response to carboplatin. Gene set enrichment analysis on differentially expressed proteins was performed to identify biologically upregulated pathways. Results: We found differentially expressed proteins related to response to carboplatin in treatment naive tumors. Biologically upregulated pathways have been identified in patients with high tumor shrinkage after treatment with carboplatin. The results demonstrate the importance of the pre-treatment protein landscape in prediction of benefit to carboplatin. Studies regarding biological mechanisms related to response in both TN and HR+ breast cancer, and differences among these subtypes, are ongoing. Conclusion: Our study provides new insight into the mechanisms of response and resistance to carboplatin treatment in the neoadjuvant setting for HER2-negative breast cancer patients. The results support further studies on the protein landscape of tumors as a complementary method to gene expression analysis to unravel the molecular mechanisms of sensitivity and resistance to treatment in breast cancer. Citation Format: Maria Aanesland Dahle, Eivind Valen Egeland, Lina Prasmickaite, Ole Christian Lingjærde, Hege Russnes, Øystein Garred, Marianne L. Smebye, Helle Skjerven, Ellen Schlichting, Bjørn Naume, Gunhild Mælandsmo, Olav Engebraaten, Mads H. Haugen. Pre-treatment protein landscape in HER2-negative breast cancer treated with carboplatin in a neoadjuvant chemotherapy setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2170.

Assessment of drug sensitivity in tumor tissue ex vivo may significantly contribute to functional diagnostics to guide personalized treatment of cancer. Tumor organoid- and explant-cultures have become attractive tools towards this goal, although culturing conditions for breast cancer (BC) tissue have been among the most challenging to develop. Validation of possibilities to detect concordant responses in individual tumors and their respective cultures ex vivo is still needed. Here we employed BC patient-derived xenografts (PDXs) with distinct drug sensitivity, to evaluate different conditions for tissue dissociation, culturing and monitoring of treatment efficacy ex vivo, aiming to recapitulate the in vivo drug responses. The common challenge of discriminating between tumor and normal cells in the cultured tissue was also addressed. Following conventional enzymatic dissociation of BC tissue, the tumor cells stayed within the non-disrupted tissue fragments, while the single cells represented mostly normal host cells. By culturing such fragments as explants, viable tumor tissue could be maintained and treated ex vivo, providing representative indications on efficacy of the tested treatment. Thus, drug sensitivity profiles, including acquired chemoresistance seen in the PDXs, were recapitulated in the respective explants. To detect the concordant responses, however, the effect monitoring had to be harmonized with the characteristics of the cultured tissue. In conclusion, we present the feasibility of BC explants ex vivo to capture differences in drug sensitivity of individual tumors. The established protocols will aid in setting up an analogous platform for BC patient biopsies with the aim to facilitate functional precision medicine.

Our understanding of how speed and persistence of cell migration affects the growth rate and size of tumors remains incomplete. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation, a phenomenon often linked to alterations of cell migration. Golgi fragmentation was induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the experimentally obtained migration and invasion traits of Giantin depleted breast cancer cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.

Resistance to chemotherapy is a major clinical challenge in breast cancer (BC), and patients developing resistance need treatment alternatives. In this project we utilize patient-derived xenografts (PDXs) from triple negative BC (TNBC), as these models better reflect the human tumor complexity and heterogeneity, compared to cell line-based- and transgene animal models. An in-house established orthotopically growing PDX, MAS98.12, has gained resistance to chemotherapy due to prolonged exposure to paclitaxel (MAS98.12-PR), a microtubule-targeting chemotherapeutic agent. As the resistance was obtained in animals, we also have the paclitaxel sensitive equivalent (MAS98.12-PS). By utilizing the pair of sensitive and resistant tumors we can study the corresponding mechanism evolving in patients who initially respond to chemotherapy before resistance and disease progression. This model will aid in identifying key mechanisms of survival in the resistant tumors, and eventually in identifying targets for use as novel therapeutic opportunities in TNBC patients resistant to standard therapy. Finally, we have samples collected early and late after resistance development, allowing us to characterize mechanisms involved in stepwise progression of paclitaxel resistance. We are currently performing molecular profiling to reveal the mechanisms involved in development of resistance. Data from bulk RNAseq, immune profiling of myeloid cells, exome sequencing, secretome profiling and reverse-phase protein array (RPPA) allow for characterizing the isogenic pair of tumors on multiple levels. Preliminary observations include aberrant expression of ABCB1/MDR1, normally involved in translocating drugs across membranes, and previously described as a mechanism involved in pertaining resistance in tumor cells. As multiple attempts to target MDR1 itself has failed, we hypothesize that our PS and PR pair will identify vulnerable/targetable signaling axis in the resistant tumors. Additionally, both PDX models have been assessed for their sensitivity to various chemo-/targeted-drugs and their combinations, revealing alternative means of treating the resistant tumors. Altogether, the molecular profiles and the sensitivity data will give novel knowledge and treatment options for patients failing to respond to standard chemotherapy. Citation Format: Eivind Valen Egeland, Kotryna Seip, Geir Frode Øy, Eleni Skourti, Solveig J Pettersen, Siri Juell, Mads Haugland Haugen, Olav Engebraaten, Lina Prasmickaite, Gunhild M Maelandsmo. Chemoresistant patient-derived xenografts to identify treatment options in breast cancer patients not responding to standard chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4675.

More than half of metastatic melanoma patients receiving standard therapy fail to achieve a long-term survival due to primary and/or acquired resistance. Tumor cell ability to switch from epithelial to a more aggressive mesenchymal phenotype, attributed with AXLhigh molecular profile in melanoma, has been recently linked to such event, limiting treatment efficacy. In the current study, we investigated the therapeutic potential of the AXL inhibitor (AXLi) BGB324 alone or in combination with the clinically relevant BRAF inhibitor (BRAFi) vemurafenib. Firstly, AXL was shown to be expressed in majority of melanoma lymph node metastases. When treated ex vivo, the largest reduction in cell viability was observed when the two drugs were combined. In addition, a therapeutic benefit of adding AXLi to the BRAF-targeted therapy was observed in pre-clinical AXLhigh melanoma models in vitro and in vivo. When searching for mechanistic insights, AXLi was found to potentiate BRAFi-induced apoptosis, stimulate ferroptosis and inhibit autophagy. Altogether, our findings propose AXLi as a promising treatment in combination with standard therapy to improve therapeutic outcome in metastatic melanoma.

Cellular phenotype plasticity between the epithelial and mesenchymal states has been linked to metastasis and heterogeneous responses to cancer therapy, and remains a challenge for the treatment of triple‐negative breast cancer (TNBC). Here, we used isogenic human breast epithelial cell lines, D492 and D492M, representing the epithelial and mesenchymal phenotypes, respectively. We employed a CRISPR‐Cas9 loss‐of‐function screen targeting a 2240‐gene ‘druggable genome’ to identify phenotype‐specific vulnerabilities. Cells with the epithelial phenotype were more vulnerable to the loss of genes related to EGFR‐RAS‐MAPK signaling, while the mesenchymal‐like cells had increased sensitivity to knockout of G2‐M cell cycle regulators. Furthermore, we discovered knockouts that sensitize to the mTOR inhibitor everolimus and the chemotherapeutic drug fluorouracil in a phenotype‐specific manner. Specifically, loss of EGFR and fatty acid synthase (FASN) increased the effectiveness of the drugs in the epithelial and mesenchymal phenotypes, respectively. These phenotype‐associated genetic vulnerabilities were confirmed using targeted inhibitors of EGFR (gefitinib), G2‐M transition (STLC), and FASN (Fasnall). In conclusion, a CRISPR‐Cas9 loss‐of‐function screen enables the identification of phenotype‐specific genetic vulnerabilities that can pinpoint actionable targets and promising therapeutic combinations.

In this study, we probed the importance of O-GlcNAc transferase (OGT) activity for the survival of tamoxifen-sensitive (TamS) and tamoxifen-resistant (TamR) breast cancer cells. Tamoxifen is an antagonist of estrogen receptor (ERα), a transcription factor expressed in over 50% of breast cancers. ERα-positive breast cancers are successfully treated with tamoxifen; however, a significant number of patients develop tamoxifen-resistant disease. We show that in vitro development of tamoxifen-resistance is associated with increased sensitivity to the OGT small molecule inhibitor OSMI-1. Global transcriptome profiling revealed that TamS cells adapt to OSMI-1 treatment by increasing the expression of histone genes. This is known to mediate chromatin compaction. In contrast, TamR cells respond to OGT inhibition by activating the unfolded protein response and by significantly increasing ERRFI1 expression. ERRFI1 is an endogenous inhibitor of ERBB-signaling, which is a known driver of tamoxifen-resistance. We show that ERRFI1 is selectively downregulated in ERα-positive breast cancers and breast cancers driven by ERBB2. This likely occurs via promoter methylation. Finally, we show that increased ERRFI1 expression is associated with extended survival in patients with ERα-positive tumors (p = 9.2e−8). In summary, we show that tamoxifen-resistance is associated with sensitivity to OSMI-1, and propose that this is explained in part through an epigenetic activation of the tumor-suppressor ERRFI1 in response to OSMI-1 treatment.

Triple-negative breast cancer (TNBC) and malignant melanoma are highly aggressive cancers that widely express the cell surface chondroitin sulfate proteoglycan 4 (CSPG4/NG2). CSPG4 plays an important role in tumor cell growth and survival and promotes chemo- and radiotherapy resistance, suggesting that CSPG4 is an attractive target in cancer therapy. In the present work, we applied the drug delivery technology photochemical internalization (PCI) in combination with the novel CSPG4-targeting immunotoxin 225.28-saporin as an efficient and specific strategy to kill aggressive TNBC and amelanotic melanoma cells. Light-activation of the clinically relevant photosensitizer TPCS2a (fimaporfin) and 225.28-saporin was found to act in a synergistic manner, and was superior to both PCI of saporin and PCI-no-drug (TPCS2a + light only) in three TNBC cell lines (MDA-MB-231, MDA-MB-435 and SUM149) and two BRAFV600E mutated malignant melanoma cell lines (Melmet 1 and Melmet 5). The cytotoxic effect was highly dependent on the light dose and expression of CSPG4 since no enhanced cytotoxicity of PCI of 225.28-saporin compared to PCI of saporin was observed in the CSPG4-negative MCF-7 cells. The PCI of a smaller, and clinically relevant CSPG4-targeting toxin (scFvMEL-rGel) validated the CSPG4-targeting concept in vitro and induced a strong inhibition of tumor growth in the amelanotic melanoma xenograft A-375 model. In conclusion, the combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.

Abstract Recently the biological and therapeutic perspective on cancer has evolved from focusing on tumor cells only, to include the complex impact of the tumor microenvironment (TME). The TME appears to have a strong influence on both tumor progression and response to treatment. In breast cancer, and in particular the triple-negative subgroup, patient outcome together with the therapeutic response are strongly linked to the tumor's inflammatory profile: presence of tumor-infiltrating leukocytes and their regulatory cytokine. Tumor-associated macrophages (TAMs) are among the most abundant immune cells in the TME, and expression of such cells has been strongly correlated with both poor outcome and development of resistance. In this study we explored the effect of the pro-metastatic, inflammation-associated, TME factor S100A4 on breast cancer cells (BCCs) of different subtypes, and their further interactions with inflammatory cells. We show that S100A4 activates BCCs, stimulating secretion of pro-inflammatory cytokines, which, in turn, promotes monocyte conversion into TAM-like cells. This was in particular prominent for cytokines secreted from basal-like BCCs. The TAM-like cells possess pro-tumorigenic activities, including increased resistance and ability to migrate. In conclusion, we demonstrate that S100A4 instigate an inflammatory microenvironment, involving a network of cytokines and TAMs, which is particularly pronounced in basal-like BC and could facilitate aggressive phenotypes of this subtype. Citation Format: Lina Prasmickaite, Ellen M. Tenstad, Eivind Valen Egeland, Solveig Pettersen, Shakila Jabeen, Silje Nord, Mads Haugland Haugen, Siri Juell, Tove Øyjord, Olav Engebråten, Gunhild Mari Mælandsmo. Basal-like breast cancer engages tumor-supportive macrophages via cytokines triggered by extracellular S100A4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 114.

The tumor microenvironment ( TME ) may influence both cancer progression and therapeutic response. In breast cancer, particularly in the aggressive triple‐negative/basal‐like subgroup, patient outcome is strongly associated with the tumor's inflammatory profile. Tumor‐associated macrophages ( TAM s) are among the most abundant immune cells in the TME , shown to be linked to poor prognosis and therapeutic resistance. In this study, we investigated the effect of the metastasis‐ and inflammation‐associated microenvironmental factor S100A4 on breast cancer cells ( BCC s) of different subtypes and explored their further interactions with myeloid cells. We demonstrated that extracellular S100A4 activates BCC s, particularly the basal‐like subtype, to elevate secretion of pro‐inflammatory cytokines. The secreted factors promoted conversion of monocytes to TAM ‐like cells that exhibited protumorigenic activities: stimulated epithelial–mesenchymal transition, proliferation, chemoresistance, and motility in cancer cells. In conclusion, we have shown that extracellular S100A4 instigates a tumor‐supportive microenvironment, involving a network of cytokines and TAM ‐like cells, which was particularly characteristic for basal‐like BCC s and potentiated their aggressive properties. The S100A4– BCC – TAM interaction cascade could be an important contributor to the aggressive behavior of this subtype and should be further explored for therapeutic targeting.

Cancer cells' phenotypic plasticity, promoted by stromal cells, contributes to intra-tumoral heterogeneity and affects response to therapy. We have disclosed an association between fibroblast-stimulated phenotype switching and resistance to the clinically used BRAF inhibitor (BRAFi) vemurafenib in malignant melanoma, revealing a challenge in targeting the fibroblast-induced phenotype. Here we compared molecular features and drug sensitivity in melanoma cells grown as co-cultures with fibroblasts versus mono-cultures. In the presence of fibroblasts, melanoma cells switched to the dedifferentiated, mesenchymal-like, inflammatory phenotype that showed reduced sensitivity to the most of 275 tested cancer drugs. Fibroblasts, however, sensitized melanoma cells to PI3K inhibitors (PI3Ki) and particularly the inhibitor of GSK3, AR-A014418 (GSK3i), that showed superior efficacy in co-cultures. The proteome changes induced by the BRAFi + GSK3i combination mimicked changes induced by BRAFi in mono-cultures, and GSK3i in co-cultures. This suggests that the single drug drives the response to the combination treatment, depending on fibroblast presence or absence, consequently, phenotype. We propose that the BRAFi and GSK3i (or PI3Ki) combination exemplifies phenotype-specific combinatorial treatment that should be beneficial in phenotypically heterogeneous tumors rich in stromal interactions.

Abstract Metastatic melanoma is notorious for the ability to change its phenotype in response to signals from the microenvironment, which might influence how melanoma responds to therapy. We have disclosed an association between fibroblast-induced phenotypic alterations in melanoma and resistance to the mutated BRAF inhibitor vemurafenib (BRAFi). This signifies the need to find other targets than BRAF to eliminate stroma-influenced melanoma cells. To approach this challenge, we performed proteomic analysis and cancer drug sensitivity screening, comparing fibroblast-supported versus non-supported melanoma cells. We showed that the effect of fibroblasts was critically dependent on cell-cell proximity, where melanoma cells get trapped in a fibronectin network, produced by adjacent fibroblasts. In such environment, melanoma cells down-regulate melanocytic programs (MITF-driven), gain mesenchymal features (AXL, PDGFR, fibronectin) and activate stress/inflammatory-response signaling pathways (JNK and STAT3). Altogether, this indicates fibroblast-induced melanoma transition to a de-differentiated, mesenchymal-like, pro-inflammatory phenotype. Melanoma cells with such phenotype were less responsive to BRAF/MAPK inhibitors and a number of other targeted drugs. However, they showed enhanced sensitivity to PI3K/mTOR inhibitors and, particularly, an inhibitor of GSK3b, stimulating Wnt/b-catenin signaling. Further, we employed flow cytometry to measure the levels of Ki67 and pS6 in single melanoma cells upon different conditions/treatments. Such analysis allowed discrimination of cell subpopulations representing a proliferative and a quiescent cellular state, and nicely reflected the influence of the tested drugs in the presence or absence of fibroblasts. We observed a subpopulation of proliferative pS6high/Ki67high melanoma cells, which remained after treatment with BRAFi if fibroblasts were present. This, fibroblast-protected BRAFi-resistant cell subpopulation, could be reduced/eliminated by PI3K or GSK3b inhibitors, verifying PI3K/GSK3 as potential targets in fibroblast-rich tumors. Currently, we are using mass cytometry (CyTOF) to further characterize cell subpopulations with respect to multiple markers related to cell signaling and immune interactions. Preliminary results indicate that not only signaling protein levels, but also levels of immunoregulatory proteins are altered in melanoma cells that get support from the fibroblasts. In conclusion, we demonstrate fibroblast-induced melanoma switching to a mesenchymal-like pro-inflammatory phenotype, which favors melanoma resistance to BRAF inhibitors, but sensitizes to inhibitors of PI3K/mTOR-associated signaling. CyTOF-analysis of complex tumor-stroma cell systems is used to search for additional strategies to target stroma-supported melanoma cells, either at the level of signaling, or immune interactions. Citation Format: Kotryna Seip, Marco V. Haselager, Kjetil Jørgensen, Marco Albrecht, Mads H. Haugen, Eivind Valen Egeland, Philippe Lucarelli, Thomas Sauter, Olav Engebraaten, Gunhild M. Mælandsmo, Lina Prasmickaite. Targetable nodes in fibroblast-supported melanoma cells that show resistance to BRAF inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4329. doi:10.1158/1538-7445.AM2017-4329