Recent studies have shown that the tumor extracellular matrix (ECM) associates with immunosuppression, and that targeting the ECM can improve immune infiltration and responsiveness to immunotherapy. A question that remains unresolved is whether the ECM directly educates the immune phenotypes seen in tumors. Here, we identify a tumor-associated macrophage (TAM) population associated with poor prognosis, interruption of the cancer immunity cycle, and tumor ECM composition. To investigate whether the ECM was capable of generating this TAM phenotype, we developed a decellularized tissue model that retains the native ECM architecture and composition. Macrophages cultured on decellularized ovarian metastasis shared transcriptional profiles with the TAMs found in human tissue. ECM-educated macrophages have a tissue-remodeling and immunoregulatory phenotype, inducing altered T cell marker expression and proliferation. We conclude that the tumor ECM directly educates this macrophage population found in cancer tissues. Therefore, current and emerging cancer therapies that target the tumor ECM may be tailored to improve macrophage phenotype and their downstream regulation of immunity.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. CRC develops in a complex tumour microenvironment (TME) with both mesenchymal stromal cells (MSCs) and immune infiltrate, shown to alter disease progression and treatment response. We hypothesised that an accessible, affordable model of CRC that combines multiple cell types will improve research translation to the clinic and enable the identification of novel therapeutic targets. A viable gelatine-methacrloyl-based hydrogel culture system that incorporates CRC cells with MSCs and a monocyte cell line was developed. Gels were analysed on day 10 by PCR, cytokine array, microscopy and flow cytometry. The addition of stromal cells increased transcription of matrix remodelling proteins FN1 and MMP9, induced release of tumour-promoting immune molecules MIF, Serpin E1, CXCL1, IL-8 and CXCL12 and altered cancer cell expression of immunotherapeutic targets EGFR, CD47 and PD-L1. Treatment with PD153035, an EGFR inhibitor, revealed altered CRC expression of PD-L1 but only in gels lacking MSCs. We established a viable 3D model of CRC that combined cancer cells, MSCs and monocytic cells that can be used to research the role the stroma plays in the TME, identify novel therapeutic targets and improve the transitional efficacy of therapies.

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine.

Pancreatic cancer tissues are made of different cell populations surrounded by a dense extracellular matrix. The stiff cancerous matrix impairs the diffusion of cytotoxic drugs, contributing to poor outcomes for patients. Matrix-targeting therapies normalise the extracellular matrix and, therefore, cell-matrix interactions. However, our knowledge of how matrix stiffness influences cancer cell and transcriptional dynamics and responses to anti-cancer compounds is incomplete. Here we developed a 3D cancer model to replicate the stiffness of patient-derived tissues and evaluated the effects of matrix-targeting compounds. Transcriptomic analyses showed that matrix stiffness regulated matrisome-related genes, cytokines and chemokines. The incusion of stromal cells further increased the mechanical properties of our 3D cancer model. Treatment with the ROCK inhibitor fasudil induced matrix softening and improved sensitivity of cancer and stromal cells to cytotoxic treatment, whereas inhibition of matrix metalloproteinases disrupted cancer cell invasion. Our results indicate that matrix stiffness impacts cancer cell profiles and targeting the cancerous matrix may lead to improved combination therapies for pancreatic cancer. STATEMENT OF SIGNIFICANCE: Pancreatic cancers have a dense extracellular matrix that drives resistance to therapy and disease progression, yet the mechanistic links between matrix stiffness and cancer cell behaviour are unclear. We engineered a 3D cancer model based on mechanical profiling of patient tissues to investigate how matrix stiffness influences gene expression and drug response. We found that increased stiffness regulates matrisome and inflammatory genes, and that stromal-induced stiffening affects sensitivity to matrix-targeting and cytotoxic drugs. The inhibition of Rho-kinase signalling enhanced the efficacy of cytotoxic drugs. These findings establish matrix stiffness as a key factor in tumour biology and therapy response, supporting the use of our preclinical model to guide new combination treatments for pancreatic cancer, aimed at improving outcomes for patients.

Introduction: More than 150,000 women die worldwide every year of Triple-Negative Breast Cancer (TNBC). There are a range of treatment options, but no good way to match patients to their optimal treatment. For most newly diagnosed patients with early TNBC, the current standard of care is neoadjuvant chemo/immunotherapy before surgery, with patients who achieve a pathological complete response (pCR) having a better prognosis. We have developed a Functional Precision Medicine (FPM) test that uses a fresh biopsy, dissociates the cells, embeds them in a 3D hydrogel matrix, cultures them in a microfluidics device and tests them against a range of systemic therapies, while using a computer vision pipeline to measure responses to therapies ex vivo. Methods: We designed and conducted an observational multi-centre clinical trial to assess the feasibility of using our FPM assay in patients with newly diagnosed TNBC undergoing neoadjuvant therapy. Patients underwent an additional core needle biopsy followed by systemic therapy as part of routine care. We assessed the response in our assay against whether patients achieved pCR or not at the time of definitive surgery, and calculated Receiver-Operating Characteristic curves (ROC) to optimize cut-offs. In patients who did not achieve pCR, we explored whether there were other regimens that had a better in-assay performance. Results: In cohort A, we recruited 34 patients, of whom 12 are evaluable as of 31st July 2024 All were female. Nine patients achieved a pCR. Specificity was 100%, sensitivity 78%, p = 0.0455 and the AUC for the ROC for predicting pCR vs. non-pCR was 0.78. In the 3 patients who did not achieve a pCR, one patient had a regimen that performed better in assay than the treatment they received, and where the response was greater than the cut-off that predicted pCR in other patients. Conclusion: We have presented interim results from a novel FPM assay in patients with early stage TNBC. Our test demonstrates good performance in predicting pCR. The trial continues to accrue data, and Cohort B continues to recruit (PEAR-TNBC; NCT05435352 ). CoI statement: Williams, Peerani, Tham, Iori, de Fraine, Loughrey, Kaffa, Richardson, Liberal, Velentza-Almpani, Wiskerke, Sangkolah, Crawley, Kearney, Bah, Ranatunga are employees of Pear Bio, with salary, stock options and IP. Hall has received honoraria from Pfizer, Eisai, MSD, Seagen, Exact Sciences, Gilead, AstraZeneca and conference expenses from Lilly and Novartis. Williams has research funding or agreements from Cancer Research UK, Breast Cancer Now, The Brain Tumour Charity, Brain Tumour Research and Novocure. Tasoulis has received honoraria from the BMJ and Company: BMJ and IntegraConnect Kirwan reports no Conflict of Interest

Recent studies have shown the tumor extracellular matrix (ECM) associates with immunosuppression, and that targeting the ECM can improve immune infiltration and immunotherapy response. A question that remains is whether the ECM is directly educating the immune phenotypes seen in cancer. We identified a tumor-associated macrophage (TAM) population correlated with poor prognosis, interruption of the cancer immunity cycle, and tumor ECM composition. To investigate whether ECM was capable of generating the TAM phenotype seen, we developed a decellularized tissue model that retains the native ECM architecture and composition. Macrophages cultured on decellularized ovarian metastasis shared transcriptional profiles with the TAMs found in human tissues. ECM educated macrophages have a tissue remodeling and immunoregulatory phenotype, inducing altered T cell function. We conclude that the tumor ECM is directly educating this macrophage population found in cancer tissues. Therefore, current and emerging cancer therapies that target the tumor ECM may be tailored to improve macrophage phenotype and their downstream regulation of immunity.