The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.

The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.

Just as Koch’s postulates formed the foundation of early infectious disease study, Stanley Falkow’s molecular Koch’s postulates define best practice in determining whether a specific gene contributes to virulence of a pathogen. Fundamentally, these molecular postulates state that if a gene is involved in virulence, its removal will compromise virulence. Likewise, its reintroduction should restore virulence to the mutant. These approaches are widely employed in Cryptococcus neoformans, where gene deletion via biolistic transformation is a well-established technique. However, the complementation of these mutants is less straightforward. Currently, one of three approaches will be taken: the gene is reintroduced at the original locus, the gene is reintroduced into a random site in the genome, or the mutant is not complemented at all. Depending on which approach is utilized, the mutant may be complemented but other genes are potentially disrupted in the process. To counter the drawbacks of the current approaches to complementation we have created a new tool to assist in this key step in the study of a gene’s role in virulence. We have identified and characterized a small gene-free region in the C. neoformans genome dubbed the “safe haven”, and constructed a plasmid vector that targets DNA constructs to this preselected site. The plasmid vector integrates with high frequency, effectively complementing a mutant strain without disrupting adjacent genes. qRT-PCR of the flanking genes on either side of the safe haven site following integration of the targeting vector revealed no changes in their expression, and no secondary phenotypes were observed in a range of phenotypic assays including an intranasal murine infection model. Combined, these data confirm that we have successfully created a much-needed molecular resource for the Cryptococcus community, enabling the reliable fulfillment of the molecular Koch’s postulates.

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. Significance: This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC.

The lysyl oxidase (LOX) family of enzymes are a major driver in the biogenesis of desmoplastic matrix at the primary tumour and secondary metastatic sites. With the increasing interest in and development of anti-stromal therapies aimed at improving clinical outcomes of cancer patients, the Lox family has emerged as a potentially powerful clinical target. This review examines how lysyl oxidase family dysregulation in solid cancers contributes to disease progression and poor patient outcomes, as well as an evaluation of the preclinical landscape of LOX family targeting therapeutics. We also discuss the suitability of the LOX family as a diagnostic and/or prognostic marker in solid tumours.