On-treatment biomarkers for assessing the response to immune-oncology (I/O) therapy have a better predictive value than baseline markers, prior to treatment1. The Farcast TruTumor histoculture platform that preserves the near native tumor microenvironment has the potential to identify such biomarkers post treatment with I/O therapy2. Accessing fresh tumor sample is often challenging, limiting the clinical utility of such ex-vivo platforms. To address this limitation, we assessed exosomes from explant culture supernatants post-treatment, for identifying response biomarkers in the culture supernatant that mimic the TME response and will seek to validate these in exosomes from matched patient plasma. Head and Neck Squamous Cell Carcinoma (HNSCC) surgical excess samples along with matched blood were used for this study. Explants generated from these samples treated with anti-PD1, Nivolumab (132µg/ml) for 72 hours, and response evaluated using flow cytometry and cytokine release analysis. Exosomes were isolated from culture supernatant and plasma using a combination of precipitation and CD63/81/9 coated magnetic bead-based purification. Exosome size was verified using nano particle tracking and further characterized using multicolor flowcytometry analysis. We observed similar distribution patterns of exosomes from the culture supernatant and matched plasma, with highest proportion of CD63+ exosomes. We evaluated exosomal composition of two samples (S1 and S2) that responded differentially to anti-PD1 treatment. S1 displayed strong anti-PD1 treatment driven T cell reinvigoration phenotype along with tumor cytolytic activity, while S2 did not. There was a treatment induced increase in T cell derived exosome (CD3+, TCex) and Interferon gamma (IFNg) positive TCex in S1 but a decrease of both in S2, mirroring the strong response phenotype in S1. We observed a decrease in PD1/PDL1 ratio in exosomes from the culture supernatant of S1 (∼1.5 fold) indicative of decreased exhaustion. This correlated with increase in IFNg secretion on treatment. The PD1/PDL1 ratio remained unchanged in S2. Additionally, TCex/ M2 like macrophage derived exosome (CD206+, MPex) ratio was higher in S1 (1.8) compared to S2 (0.7) upon treatment, indicative of an M2 like macrophage driven immunosuppressive TME that explained the non-responsive phenotype in S2. A similar picture emerged on immune cell profiling, wherein, on treatment S1 had a higher T cell/ M2 macrophage ratio (18.4) compared to S2 (1.9). The data presented from the TruTumor ex vivo platform illustrates good concordance between treatment induced changes in immune cell derived exosome composition and intra-tumoral immune cell profile. This platform presents a unique opportunity for identification of exosomal biomarkers that could be potentially tracked in periphery. 1. Chen, P.L et al. Cancer Discov. 6, 827–837 (2016) 2. Basak, NP et al. Nat Commun 15, 1585 (2024). Citation Format: Kowshik Jaganathan, Moumita Nath, Biswajit Das, Méhul Kapur, Christopher Davitt, Ritu Malhotra, Mohit Malhotra, Nandini Pal Basak, Satish Sankaran. Identification of tumor microenvironment informed exosome biomarkers for predicting response to anti-PD1 therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B030.
Kumamolisin from Bacillus sp. MN 32 (produced by E. coli B/BL21 background) is an acid active sedolisin protease developed for food uses; its systemic safety was assessed using GLP (Good-laboratory-practice) in vitro genotoxicity assays and repeated dose oral studies. A bacterial reverse mutation assay (OECD TG 471; Salmonella TA98, TA100, TA102, TA1535, TA1537; plate incorporation and preincubation; +/-S9) showed no concentration related increases in revertants up to 5000 ug total organic solids (TOS)/plate. A mammalian cell micronucleus test (OECD TG 487; human lymphocytes; 4 h +/-S9 and 24 h -S9) showed no significant increases in micronucleated binucleated cells at 1250-5000 ug TOS/mL, with cytotoxicity <= 21.6% by CBPI (Cytokinesis-Block Proliferation Index). In 28 day and 90 day repeated dose toxicity studies Sprague Dawley rat studies (OECD TG 407/408; n = 10/sex/group) at 0, 500, 1000, and 2000 mg TOS/kg bw/day by gavage (10 mL/kg/day), survival was 100% and in life observations, ophthalmology, neurobehavior, urinalysis, hematology, coagulation, and clinical chemistry were unremarkable; isolated statistical differences (e.g., male total protein, BUN, glucose, LDL; calcium; female phosphorus) were marginal, within historical ranges, non monotonic, and lacked histopathology correlates. Organ weight differences were single sex and non dose dependent. No test item related lesions were detected. The NOAEL was 2000 mg TOS/kg bw/day (highest dose tested) in both the 28-day and 90-day studies, supporting oral safety for food use exposure. ### Competing Interest Statement Study is funded and supported by Amplifye Inc. Amplifye Inc.
Immune checkpoint inhibition (ICI) holds great promise for triple-negative breast cancer (TNBC), while it shows limited response hormone receptor positive (ER/PR+) breast cancer (CaBr). We employed the Farcast CaBr TruTumor histoculture platform, that preserves the native tumor microenvironment (TME), to study the role of tumor resident immune cell types in determining T cell activation levels on ICI treatment, in the two CaBr sub-types. Freshly resected tumor tissue samples along with matched blood were collected from consented patients. Tumor explants were generated and distributed into arms and cultured for 72 h. Media was replenished every 24 h and the supernatant was stored. The response to stimulation with anti-CD3 (100 ng/mL) + Interleukin-2 (IL-2, 100 IU/mL) and treatment with Nivolumab (132 µg/mL) was evaluated using cytokine release and flow cytometry based immune profiling. CaBr (n=115) samples had lower immune component than head and neck squamous cell carcinoma (n=113) (p<0.0001) but similar to ovarian (n=21) and renal cell (n=52) cancer prior to treatment with ICI. Amongst the two sub-types, immune component in TNBC (n=43) was higher than ER/PR+ subtypes (n=46) (p<0.05). TNBC (n=10) showed a greater proportion of lymphocyte (58.46%) compared to myeloid (39.21%) compartment. This bias was not observed in ER/PR+ samples (n=12). Anti-CD3+IL2 stimulation showed similar response between the two CaBr sub-types. Upon treatment with Nivolumab, 2 out of 4 TNBC samples exhibited a response phenotype, with >1.6-fold increase in CD8+GzmB+ cells and interferon-gamma (IFN-γ) release. In contrast, only 1 out of 4 ER/PR+ CaBr samples showed a modest increase of 1.3 fold for CD8+GzmB+ cells with no detectable IFN-γ release. To understand the basis for the differential response in the two sub-types, we studied in detail, one ER/PR+ (H1) and two TNBC (T1 and T2) samples, displaying varying levels of response. T2 and H1 did not show a Nivolumab response phenotype, whereas T1 demonstrated a strong T-cell reinvigoration and tumor cytotoxicity. Interestingly, all three samples showed anti-CD3+IL2 stimulation driven T cell response. tSNE analysis of CTLs in the control arm showed two distinct sub-populations of exhausted CTLs (CD8+PD1+). Population1 (Pop1) was Granzyme B-positive, while population2 (Pop2) was not. Upon anti-CD3 stimulation, Pop1 showed further increase of activation whereas Pop2 did not, indicative of Pop2 being an irreversibly exhausted T cell population. T2 notably had lower Pop1 and Pop2 CTLs, along with highest proportion of monocytes across all three samples pointing towards an immunosuppressive TME. H1 mainly contained over-exhausted Pop2 and negligible Pop1 CTLs. Interplay between different TME immune sub-types, thus influence response to Nivolumab in CaBr. This is effectively captured by the TruTumor platform. Mouniss MM, Biswajit Das, Kowshik Jaganathan, Syamkumar V, Moumita Nath, Chandan Bhowal, Dharanidharan M, Saikrishna S, Abdul Haseeb, Pallavi R, Kubera Chandran, Rajashekar M, Oliyarasi M, Méhul Kapur, Jayaprakash C, Venkatesh T, Ganesh MS, Amritha Prabha, Prakash BV, Ravi Krishnappa, Upendra K, Ritu Malhotra, Govindaraj K, Pavithira ., Mohit Malhotra, Nandini Pal Basak, Satish Sankaran. Differential T cell response to anti-PD1 in breast cancer Sub-types is driven by activity of intra-tumoral immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5807.
Whilst the advent of Immune Checkpoint Blockade has revolutionized the management of cancer, a significant proportion of patients have limited or absent response to these therapies. A key cause of this immune insensitivity is the hostile solid tumor microenvironment (TME) dominated by immunosuppressive myeloid cells. We previously identified the acid sensing G protein coupled receptor (GPCR), GPR65, as a primary determinant of these suppressive cells. In mice, genetic deletion of Gpr65 or oral administration of small molecule GPR65 inhibitors in vivo causes a profound repolarization of immunosuppressive tumor associated macrophages, an increase in infiltrating effector cells and potent anti tumor effects in syngeneic models. In TCGA data, across all tumors, patients homozygous for a hypomorphic coding variant in GPR65 (I231L) show increased overall survival, providing compelling genetic evidence of the clinical potential of GPR65 inhibition. To further explore the translational potential of GPR65 we employed a range of techniques to define the human biology of this receptor in different contexts. At the mechanistic level, single cell RNA sequencing (scRNAseq) of human PBMCs obtained from healthy donors demonstrated a pronounced effect of low pH on the myeloid compartment, with a clear polarization of these cells toward an immunosuppressive character and modulation of GPR65 expression. In parallel, pharmacological inhibition of GPR65 in human monocyte derived macrophages exposed to low pH demonstrated that equivalent gene expression changes are primarily due to GPR65 activation. To examine the relevance of these findings to the intact acidic human TME, we performed studies in fresh primary human tumor histocultures from clear cell renal cell carcinoma (ccRCC) patients with immunohistochemically confirmed high macrophage infiltration and carbonic anhydrase 9 (CA9) expression. In these cultures, GPR65 inhibition caused a dose dependent suppression of a geneset closely overlapping with that modulated by GPR65 in primary macrophages. Furthermore, we observed a marked decrease in immune suppressive IL10 secretion with coincident elevation of specific proinflammatory chemokines. Consistent with these findings, in vivo administration of a small molecule GPR65 inhibitor elicited similar changes in human CA9 expressing RCC PDX tumors implanted in myeloid boosted CD34+ stem cell engrafted NCG mice. In summary, inhibition of GPR65 provides a unique and genetically validated approach to favorably modify the immunosupressive TME with features highly conserved between mouse and human contexts. We propose that GPR65 inhibition holds significant clinical promise, with specific evidence around ccRCC as a potential standout indication. Citation Format: Barbara Cipriani, Alastair Corbin, David Miller, Alan Naylor, Faraz Khan, Gavin Milne, Barbara Young, Rupert Satchell, Sourav Sarkar, Mussa Quareshy, Anastasia Nika, Preeti Singh, Gavin Knox, Darryl Turner, Satish Sankaran, Nandini Pal Basak, Toszka Bohn, Tobia Bopp, Surya Koturan, Bo Sun, Benjamin Fairfax, Tom McCarthy, Stuart Hughes. The translational biology of small molecule GPR65 inhibitors: shared effects between mouse models and human primary tumors highlight the unique transformative potential of targeting a genetically validated innate immune checkpoint [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 668.
Abstract Tyrosine Kinase Inhibitors (TKIs) and Immune checkpoint inhibitors (ICIs) are used in first line treatment of clear cell renal cell carcinoma (ccRCC). Identifying patients who truly benefit from these treatments remains a challenge. Developing newer and better therapy options that fail in the clinical phase is yet another unmet need. Both these limitations could be addressed by employing testing platforms that best capture the heterogeneity and complexity of the tumor within the patient. FarcastTM TruTumor is a near native human histo-culture platform which retains the tumor and stroma along with the intra-tumoral immune compartment post culture that holds promise to improve treatment outcomes in patients. In this study, we developed a ccRCC platform using surgical excess specimens from consented patients and compared its immune profile with the head and neck squamous cell carcinoma (HNSCC) platform. ccRCC samples (n=10) were processed to generate explants and cultured for 72h and interrogated for response to treatment with Sunitinib (TKI:27.7ng/ml) or Nivolumab (anti-PD1:132µg/ml). On comparison of immune profiles between HNSCC and ccRCC at baseline, we observed a significantly lower total immune content in ccRCC. ccRCC showed comparatively higher variation in myeloid (70%CV) and lymphoid compartments (30%CV) as compared to HNSCC. Live tumor and immune cell population was found to be well preserved post-culture in ccRCC. Presence of various T cell sub-populations, monocyte and macrophage population post culture were confirmed using flowcytometry. Sunitinib treatment significantly increased caspase-3 expression (8/10 samples, p value<0.01), along with decrease in tumor content in 4/10 samples (average drop 23%). Two samples which did not respond to Sunitinib treatment showed an increase in Interferon gamma (IFNg) (Log2FC ± SD, 2.7± 0.7) and Granzyme B (Log2FC ± SD, 1.05± 0.85) secretion, in response to Nivolumab treatment. Of these, in one sample we observed an increase in CD8+ Granzyme B, CD8+ Ki67 and decrease in CD4+ FoxP3 population, with concomitant decrease in Pan CK+ population by flowcytometry. In this sample, we observed a decrease in tumor content indicating anti-tumor response to Nivolumab treatment. In summary, the TruTumor platform potentially offers personalized treatment choices to patients improving their chances of recovery. In addition, this platform could provide powerful insights into the mechanisms of a wide range of therapy molecules in development to predict their efficacy better than other simplistic and non-human testing models. Citation Format: Satish Sankaran, Kowshik Jaganathan, Gowri Shankar K, Saurabh Bhargav, Ganesh MS, Amritha Prabha, Prakash BV, Syamkumar V, Biswajit Das, Vasanth K, Manimaran A, Chandan Bhowal, Rajashekar M, Oliyarasi M, Ritu Malhotra, Govindraj K, Nandini Pal Basak. Evaluating response of renal cell carcinoma to tyrosine kinase inhibitor and immune checkpoint inhibitor using a human histo-culture platform. [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 4568.
