Summary Functional ex vivo assays using live tumor tissues have demonstrated strong predictive accuracy for response to immune checkpoint inhibitors (ICIs) but are not scalable, requiring manual processing of large resections collected at academic centers. Here, an ex vivo live tumor fragment (LTF) platform was developed using standard-of-care biopsies from 228 patients with suspected malignancy collected across prospective, multicenter observational trials and biobanks. Hierarchical clustering of ICI-mediated changes in cytokine production identified two groups: responders and nonresponders. A binary classifier (elive index) using 8 cytokines achieved an AUC of 0.99 for cluster prediction. elive index correctly predicted clinical benefit in 93% (26/28) of patients ( P = 3.2x10 -5 ) and accurately identified 83% (10/12) of objective responders. Critically, elive responders were identified among biomarker-negative patients, highlighting the platform as a scalable approach that complements existing companion diagnostics and expands the population of patients identified to benefit from ICI therapy.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, providing durable and even curative responses. However, most patients do not respond and current biomarkers (eg, programmed death 1 ligand 1 [PD-L1]), mismatch repair deficiency [dMMR]/high microsatellite instability [MSI] and tumor mutational burden) lack predictive accuracy. Ex vivo profiling of patient-derived tumor fragments shows promise as a predictive biomarker but relies on substantial surgical tissue to mitigate intra-specimen heterogeneity. Innovations are needed that address these challenges, particularly where limited tissue is available such as in core needle biopsies (CNBs). Live tumor fragments (LTFs) were generated from 59 human tumor resections and 31 CNBs from patients enrolled in observational clinical trials (ClinicalTrials.gov identifiers: NCT05478538, NCT05520099, NCT06349642) to assess cytokine induction following ICI treatment. LTFs were encapsulated in hydrogel and cultured ex vivo for up to 72 hours. A sequential treatment strategy that applies control and treatment within the same well was used with response to ICI or αCD3/αCD28 assessed using a multiplex secretome assay. Viability was assessed using established metabolic assays and dynamic optical coherence microscopy. LTFs maintained viability and retained T cells responsive to stimulation throughout ex vivo culture. Multiplex immunofluorescence and immunohistochemistry showed key components of the tumor microenvironment, including relative proportions of CD4+ and CD8+ immune cell populations, were preserved. Specimens positive for PD-L1 or dMMR/MSI-high were enriched for cytokine upregulation, including T-cell response cytokines IFNγ and CXCL10, after αPD-1 treatment. To demonstrate clinical applicability of the sequential treatment strategy, CNBs from patients with lung, gastrointestinal or kidney cancer were profiled and differential cytokine induction in response to ICI treatment was observed. The novel ex vivo platform presented is capable of detecting T-cell response to ICI treatment by using a sequential treatment strategy. This approach addresses challenges associated with cross-well heterogeneity in tissue composition and requires half as much tissue as a cross-well comparison, mitigating tissue limitations typically associated with non-surgical biopsies. Importantly, the platform is compatible with established functional assays as well as non-destructive spatial imaging, enabling researchers to characterize response to ICI longitudinally. Ongoing trials will enable clinicians to assess platform performance in predicting response to immunotherapy.

Defining the proteoform landscape of breast cancer can provide unique insights into the signaling pathways driving disease progression. While bottom-up proteomics has been utilized to profile breast cancer, it lacks the ability to capture intact proteoforms that may underpin the disease. Top-down proteomics is ideally suited to characterize intact proteoforms; however, most top-down proteomics studies have been limited to low molecular weight (MW) proteins (<50 kDa). Herein, we employed a two-dimensional (2D) liquid chromatography combining size exclusion chromatography (SEC) with reverse phase chromatography (RPC) followed by high-resolution mass spectrometry (MS) to expand the coverage for high MW proteoforms. Using this 2D-SEC-RPC-MS approach, we observed a 5-fold increase in the detection of high MW proteoforms (>50 kDa) compared to the conventional 1D-RPC-MS. SEC separation significantly enhanced the detection of high MW proteoforms (>104 kDa), including intermediate filament proteins, vimentin and keratins. Based on accurate mass measurements and MS/MS data, we identified 775 proteoforms from both TFA and HEPES extracts and detected PTMs, such as acetylation, glutathionylation, and myristoylation. Pathway analysis uncovered many proteoforms involved in processes dysregulated in cancer progression. Overall, our findings illustrate the power of top-down proteomics in defining the proteoform landscape of breast carcinoma.

Immunotherapies are highly effective treatment options for cancer; yet they do not work in all patients, and current companion diagnostic (CDx) biomarkers do not always align with response. Ex vivo profiling of live tumor samples provides the ability to measure T cell activity in response to treatment and is a potential means of improving response prediction. The limited amount of tumor tissue available from core needle biopsies (CNBs) presents a challenge when split into control and treatment groups for ex vivo profiling, as tumor heterogeneity causes increased variability and sampling errors. Elephas has developed a treatment strategy where control (IgG) and immune checkpoint inhibitor (ICI) treatments are performed sequentially on the same tissue within a single well. Changes in the rate of cytokine secretion are then compared between ICI and IgG treatment to characterize response to therapy. For these experiments, human CNBs (n>150) were collected from diverse tumor types during standard of care diagnostic procedures and sliced using a proprietary cutting instrument. Each biopsy was profiled for response to ICI using a multiplexed cytokine immunoassay. Sequential aliquots of conditioned media were collected from cultured biopsy slices at three time points: ∼4 hrs post-encapsulation with a proprietary hydrogel, following ∼16 hrs of IgG treatment and following ∼28 hrs of treatment with ICI. Cytokine results were assessed for data quality to inform inclusion/exclusion of data points for analysis/algorithm development. CDx biomarker status (PD-L1, MMR/MSI and TMB) for individual biopsies was obtained from medical records. Our results show live tumor fragments, obtained from resections where tumor tissue amount was sufficient to address inter-well heterogeneity, exhibit comparable cytokine response profiles when stimulated with CD3/CD28 at experiment start vs. following 20 hrs of IgG treatment. An algorithm to define ex vivo response to ICI treatment in CNBs was then developed using tissue viability measurements and treatment-induced changes in cytokines. Response on the Elephas platform was subsequently compared to CDx biomarker status and clinical response from patient medical records. A sequential treatment strategy for the assessment of response to immunotherapy is an effective approach to capture treatment-induced changes in cytokine response. Sequential treatment of CNBs circumvents issues of tumor heterogeneity inherent to cross-well comparison in settings of limited tissue availability. Implementation of this strategy enables the Elephas platform to differentiate potential responder and non-responder CNB specimens to immunotherapy ex vivo. Christina Scribano, Pichet Adstamongkonkul, Sean Caenepeel, Nicholas Dana, Jackie Derrick, Evan Flietner, Hilary Hernan, Christin Johnson, Nathan Marhefke, Payton McDonnell, Betsy Mulligan, Amreen Nasreen, Josh Porter, Jordyn Richardson, Sidney Schneider, Mikaela Schultz, Mike Smith, Chetan Sood, Aishwarya Sunil, Lindsey Vedder, Ellen Wargowski, Hinco Gierman, Andreas Friedl, Laura Hrycyniak, Michael Korrer, T.S. Ramasubramanian. A sequential treatment strategy for ex vivo profiling of live tumor fragments that mitigates tumor heterogeneity and tissue scarcity from core needle biopsies to characterize response to immunotherapies [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 3301.

Characterization of response to immunotherapy ex vivo requires preservation of the live tumor microenvironment (TME), including the sequestration of immune cells. Elephas has developed a methodology that incorporates proprietary cutting instruments designed to optimally fragment live tumor resections and core needle biopsies (CNBs), and a proprietary hydrogel that supports the viability of tumor fragments in ex vivo culture while sequestering T cells in their native TME. In this study, tumor specimens (human tumors and humanized PDX) were fragmented using one of two proprietary cutting instruments designed to cut tumor resections into 300 x 300 x 300μm fragments or CNBs into 300μm thick slices. Both tissue form factors were then encapsulated in a propriety hydrogel. Tissue viability was assessed via the Cell Counting Kit 8 (CCK8) assay and in some cases a lactate dehydrogenase assay. Conditioned media from culture plates harboring encapsulated fragments was collected for multiplex cytokine analysis. At the end of ex vivo experimentation tissue was paraffin embedded and formalin fixed for H&E staining and immunohistochemistry was performed to assess T cell retention. Elephas proprietary cutting instruments produced tumor fragments of consistent size and volume. These fragments retained histological characteristics of the original tumor resections from which they were derived and their encapsulation in a proprietary hydrogel improved T cell retention over 2 days of ex vivo culture. Preservation of the native TME was further demonstrated following αPD-1 treatment of tumor fragments with known CDx biomarker status where an enriched cytokine response profile was observed in biomarker positive samples. In CNBs processed on the Elephas platform, overall viability and T cell function were maintained following simulated overnight shipment at 4°C. To overcome challenges associated with the limited amount of tissue in CNBs (e.g. heterogeneity), we developed a treatment strategy where control and treatments were performed sequentially on the same tissue within a single well. Fold changes in the rate of cytokine secretion following sequential (1) control and (2) αPD-1 + STING agonist treatment exhibit improved CVs (%) relative to an inter-well treatment design (control and treatment wells). Additionally, live tumor biopsies were shown to exhibit comparable IFNγ response following treatment with αPD-1 + STING agonist at the experiment start vs. following 20 hrs of control treatment. The Elephas platform preserves overall cell viability and T cell function in the TME of CNBs ex vivo using proprietary cutting instruments and hydrogel encapsulation techniques. Using this preparation method, the Elephas platform optimally captures the heterogeneity in human tumors (resections and CNBs) while allowing for comparisons between treatment conditions. Michael Korrer, Christina Scribano, Pichet Adstamongkonkul, Todd Bakken, Torey Browning, Christian Baltes, Sean Caenepeel, Nicholas Dana, Jackie Derrick, Thomas Dietz, Evan Flietner, Hilary Hernan, Christin Johnson, Payton McDonnell, Jacob Nesemeier, Josh Porter, Abhijeet Prasad, Jordyn Richardson, Sidney Schneider, Rebecca Tarlazzi, Noah Van Der Weide, Lindsey Vedder, Ming (Veronica) Wang, Hinco Gierman, Andreas Friedl, Laura Hrycyniak, T.S. Ramasubramanian. A method for preparing tumor tissues for ex vivo culture that retains T cells within live tumor fragments while preserving tissue viability and T cell function [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 6555.

Supplementary Figure 1 from Membrane Type 1 Matrix Metalloproteinase–Mediated Stromal Syndecan-1 Shedding Stimulates Breast Carcinoma Cell Proliferation

Supplementary Figures 2-3 from Signal Transducers and Activators of Transcription Mediate Fibroblast Growth Factor–Induced Vascular Endothelial Morphogenesis

Supplementary Figure Legends 1-4 from Signal Transducers and Activators of Transcription Mediate Fibroblast Growth Factor–Induced Vascular Endothelial Morphogenesis

PDF file - 463K, Analysis of dataset GSE2990 using the RESTless Gene Signature and clustered using Euclidean distance.

Supplementary Figure Legends 1-2 from Membrane Type 1 Matrix Metalloproteinase–Mediated Stromal Syndecan-1 Shedding Stimulates Breast Carcinoma Cell Proliferation

Supplementary Figure 1 from Signal Transducers and Activators of Transcription Mediate Fibroblast Growth Factor–Induced Vascular Endothelial Morphogenesis

<p>PDF file - 29K, Tumors in GSE2990 and GSE2034 listed as RESTless or RESTfl.</p>

<p>This file contains additional technical detail (antibodies used, gating strategy for FACS) and additional experimental data to complement that shown in the main figures.</p>

The extracellular matrix (ECM) provides an architectural meshwork that surrounds and supports cells. The dysregulation of heavily post-translationally modified ECM proteins directly contributes to various diseases. Mass spectrometry (MS)-based proteomics is an ideal tool to identify ECM proteins and characterize their post-translational modifications, but ECM proteomics remains challenging owing to the extremely low solubility of the ECM. Herein, enabled by effective solubilization of ECM proteins using our recently developed photocleavable surfactant, Azo, we have developed a streamlined ECM proteomic strategy that allows fast tissue decellularization, efficient extraction and enrichment of ECM proteins, and rapid digestion prior to reversed-phase liquid chromatography (RPLC)-MS analysis. A total of 173 and 225 unique ECM proteins from mouse mammary tumors have been identified using 1D and 2D RPLC-MS/MS, respectively. Moreover, 87 (from 1DLC-MS/MS) and 229 (from 2DLC-MS/MS) post-translational modifications of ECM proteins, including glycosylation, phosphorylation, and hydroxylation, were identified and localized. This Azo-enabled ECM proteomics strategy will streamline the analysis of ECM proteins and promote the study of ECM biology.

Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan normally expressed primarily by epithelia and plasma cells, is aberrantly induced in stromal fibroblasts of breast carcinomas. Stromal fibroblast-derived Sdc1 participates in paracrine growth stimulation of breast carcinoma cells and orchestrates stromal extracellular matrix fiber alignment, thereby creating a migration and invasion-permissive microenvironment. Here, we specifically tested the role of stromal Sdc1 in metastasis.

Abstract Although antiestrogen therapies are successful in many patients with estrogen receptor alpha-positive (ERα+) breast cancer, 25% to 40% fail to respond. Although multiple mechanisms underlie evasion of these treatments, including tumor heterogeneity and drug-resistant cancer stem cells (CSC), further investigations have been limited by the paucity of preclinical ERα+ tumor models. Here, we examined a mouse model of prolactin-induced aggressive ERα+ breast cancer, which mimics the epidemiologic link between prolactin exposure and increased risk for metastatic ERα+ tumors. Like a subset of ERα+ patient cancers, the prolactin-induced adenocarcinomas contained two major tumor subpopulations that expressed markers of normal luminal and basal epithelial cells. CSC activity was distributed equally across these two tumor subpopulations. Treatment with the selective estrogen receptor downregulator (SERD), ICI 182,780 (ICI), did not slow tumor growth, but induced adaptive responses in CSC activity, increased markers of plasticity including target gene reporters of Wnt/Notch signaling and epithelial–mesenchymal transition, and increased double-positive (K8/K5) cells. In primary tumorsphere cultures, ICI stimulated CSC self-renewal and was able to overcome the dependence of self-renewal upon Wnt or Notch signaling individually, but not together. Our findings demonstrate that treatment of aggressive mixed lineage ERα+ breast cancers with a SERD does not inhibit growth, but rather evokes tumor cell plasticity and regenerative CSC activity, predicting likely negative impacts on patient tumors with these characteristics. Significance: This study suggests that treatment of a subset of ERα+ breast cancers with antiestrogen therapies may not only fail to slow growth but also promote aggressive behavior by evoking tumor cell plasticity and regenerative CSC activity. Cancer Res; 78(7); 1672–84. ©2018 AACR.

Background: Collagen fibers surrounding breast ducts may influence breast cancer progression. Syndecan-1 interacts with constituents in the extracellular matrix, including collagen fibers, and may contribute to cancer cell migration. Thus, the orientation of collagen fibers surrounding ductal carcinoma in situ (DCIS) lesions and stromal syndecan-1 expression may predict recurrence. Methods: We evaluated collagen fiber alignment and syndecan-1 expression in 227 women diagnosed with DCIS in 1995 to 2006 followed through 2014 (median, 14.5 years; range, 0.7–17.6). Stromal collagen alignment was evaluated from diagnostic tissue slides using second harmonic generation microscopy and fiber analysis software. Univariate analysis was conducted using χ2 tests and ANOVA. The association between collagen alignment z-scores, syndecan-1 staining intensity, and time to recurrence was evaluated using HRs and 95% confidence intervals (CIs). Results: Greater fiber angles surrounding DCIS lesions, but not syndecan-1 staining intensity, were related to positive HER2 (P = 0.002) status, comedo necrosis (P = 0.03), and negative estrogen receptor (P = 0.002) and progesterone receptor (P = 0.02) status. Fiber angle distributions surrounding lesions included more angles closer to 90 degrees than normal ducts (P = 0.06). Collagen alignment z-scores for DCIS lesions were positively related to recurrence (HR = 1.25; 95% CI, 0.84–1.87 for an interquartile range increase in average fiber angles). Conclusions: Although collagen alignment and stromal syndecan-1 expression did not predict recurrence, collagen fibers perpendicular to the duct perimeter were more frequent in DCIS lesions with features typical of poor prognosis. Impact: Follow-up studies are warranted to examine whether additional features of the collagen matrix may more strongly predict patient outcomes. Cancer Epidemiol Biomarkers Prev; 27(2); 138–45. ©2017 AACR.

Volume 4408–4421, 2013. We retract this publication because we recently discovered that many of the results reported in this paper can be attributed to an artifact of gene transduction rather than the transduced gene itself and that, therefore, some conclusions of this work are no longer valid. We initiated the retraction as soon as the new information became available. A replication-deficient adenovirus (pLP-Adeno-X-PRLS; Clontech) was used to overexpress glypican 1 (GPC1), and empty adenovirus (Ad-control) was used as control. Overexpression of GPC1 was confirmed by Western blotting. Recently, we performed transmission electron microscopy and detected adenoviral particles in Ad-GPC1-infected but not Ad-control-infected cells. PCR analysis confirmed the presence of E1 protein in Ad-GPC1 but not in Ad-control. Therefore, we conclude that Ad-GPC1 has acquired the E1 gene from the HEK 293 packaging cells and has become replication competent. Considering the small scale of virus production used in these experiments, this is a very unlikely event (H. Lochmüller et al., Hum Gene Ther, 5: 1485–1491, 1994; J. Zhu et al., Hum Gene Ther, 10: 113–121, 1999), which appears to have occurred. We prepared a new batch of Ad-GPC1 and repeated key experiments with U87 cells. While GPC1 overexpression induced an increase in S-phase cells, we could not reproduce the dramatic effects on bromodeoxyuridine incorporation or on the cell cycle regulators which we reported in the

The heparan sulfate proteoglycan glypican-1 (GPC1) is involved in tumorigenesis and angiogenesis and is overexpressed frequently in tumor and endothelial cells (ECs) in human gliomas. We demonstrated previously that in brain EC, GPC1 regulates mitotic cyclins and securin as well as mitosis and that GPC1 is required for progression through the cell cycle. To characterize the molecular mechanism underlying cell cycle regulation by GPC1, we systematically investigated its effects on key G1/S checkpoint regulators and on major signaling pathways reportedly activated by Dally (Division abnormally delayed) the Drosophila GPC1 homologue. We found that elevated GPC1 affected a wide range of G1/S checkpoint regulators, leading to inactivation of the G1/S checkpoint and increased S phase entry, apparently by activating the mitogen-independent Skp2 autoinduction loop. Specifically, GPC1 suppressed CDK inhibitors (CKIs), including p21, p27, p16, and p19, and the D cyclins, and induced CDK2 and Skp2. GPC1 may trigger the Skp2 autoinduction loop at least partially by suppressing p21 transcription as knockdown of p21 by RNAi can mimic the effect of GPC1 on the cell cycle regulators related to the loop. Moreover, multiple mitogenic signaling pathways, including ERK MAPK, Wnt and BMP signaling, were significantly stimulated by GPC1 as has been reported for Dally in Drosophila. Notably, the c-Myc oncoprotein, which is frequently up-regulated by both ERK and Wnt signaling and functions as a potent transcription repressor for CKIs as well as D cyclins, was also significantly induced by GPC1. These findings provide mechanistic insights into how GPC1 regulates the cell cycle and proliferation.