Probes for INS

Abstract

PURPOSE:

Four consensus molecular subtypes (CMS1-4) of colorectal cancer (CRC) were identified in primary tumors and found to be associated with distinctive biological features and clinical outcomes. Given that distant metastasis largely accounts for CRC-related mortality, we examined the molecular and clinical attributes of CMS in metastatic CRC (mCRC).

EXPERIMENTAL DESIGN:

We developed a CRC-focused Nanostring based CMS classifier that is ideally suited to interrogate archival tissues. We successfully employ this panel in the CMS classification of FFPE tissues from mCRC cohorts, one of which is comprised of paired primary tumors and metastases. Finally, we developed novel mouse implantation models to enable modelling of CRC in vivo at relevant sites.

RESULTS:

Using our classifier we find that the biological hallmarks of mCRC, including CMS, are in general highly similar to those observed in non-metastatic early stage disease. Importantly, our data demonstrate that CMS1 has the worst outcome in relapsed disease, compared to other CMS. Assigning CMS to primary tumors and their matched metastases revealed mostly concordant subtypes between primary and metastasis. Molecular analysis of matched discordant pairs revealed differences in stromal composition at each site. The development of two novel in vivo orthotopic implantation models further reinforces the notion that extrinsic factors may impact on CMS identification in matched primary and metastatic CRC.

CONCLUSION:

We describe the utility of a Nanostring panel for CMS classification of FFPE clinical samples. Our work reveals the impact of intrinsic and extrinsic factors on CRC heterogeneity during disease progression.

The decline of cognitive function occurs with aging, but the mechanisms responsible are unknown. Astrocytes instruct the formation, maturation, and elimination of synapses, and impairment of these functions has been implicated in many diseases. These findings raise the question of whether astrocyte dysfunction could contribute to cognitive decline in aging. We used the Bac-Trap method to perform RNA sequencing of astrocytes from different brain regions across the lifespan of the mouse. We found that astrocytes have region-specific transcriptional identities that change with age in a region-dependent manner. We validated our findings using fluorescence in situ hybridization and quantitative PCR. Detailed analysis of the differentially expressed genes in aging revealed that aged astrocytes take on a reactive phenotype of neuroinflammatory A1-like reactive astrocytes. Hippocampal and striatal astrocytes up-regulated a greater number of reactive astrocyte genes compared with cortical astrocytes. Moreover, aged brains formed many more A1 reactive astrocytes in response to the neuroinflammation inducer lipopolysaccharide. We found that the aging-induced up-regulation of reactive astrocyte genes was significantly reduced in mice lacking the microglial-secreted cytokines (IL-1α, TNF, and C1q) known to induce A1 reactive astrocyte formation, indicating that microglia promote astrocyte activation in aging. Since A1 reactive astrocytes lose the ability to carry out their normal functions, produce complement components, and release a toxic factor which kills neurons and oligodendrocytes, the aging-induced up-regulation of reactive genes by astrocytes could contribute to the cognitive decline in vulnerable brain regions in normal aging and contribute to the greater vulnerability of the aged brain to injury.

Prostate cancer (PCa) does not appear to respond to immune checkpoint therapies where T cell infiltration may be a key limiting factor. Here we report evidence that ablating the growth regulatory kinase Erk5 can increase T cell infiltration in an established Pten-deficient mouse model of human PCa. Mice that were doubly mutant in prostate tissue for Pten and Erk5 (prostate DKO) exhibited a markedly increased median survival with reduced tumor size and proliferation compared to control Pten-mutant mice, the latter of which exhibited increased Erk5 mRNA expression. A comparative transcriptomic analysis revealed upregulation in prostate DKO mice of the chemokines Ccl5 and Cxcl10, two potent chemoattractants for T lymphocytes. Consistent with this effect, we observed a relative increase in a predominantly CD4+ T cell infiltrate in the prostate epithelial and stroma of tumors from DKO mice. Collectively, our results offer a preclinical proof of concept for ERK5 as a target to enhance T cell infiltrates in prostate cancer, with possible implications for leveraging immune therapy in this disease.

Sipuleucel-T is the only FDA-approved immunotherapy for metastatic castration-resistant prostate cancer. The mechanism by which this treatment improves survival is not fully understood. We have previously shown that this treatment can induce the recruitment of CD4 and CD8 T cells to the tumor microenvironment. In this study, we examined the functional state of these T cells through gene expression profiling. We found that the magnitude of T cell signatures correlated with the frequency of T cells as measured by immunohistochemistry. Sipuleucel-T treatment was associated with increased expression of Th1-associated genes, but not Th2-, Th17 – or Treg-associated genes. Post-treatment tumor tissues with high CD8+T cell infiltration was associated with high levels of CXCL10 expression. On in situ hybridization, CXCL10+ cells colocalized with CD8+T cells in post-treatment prostatectomy tumor tissue. Neoadjuvant sipuleucel-T was also associated with upregulation of immune inhibitory checkpoints, including CTLA4 and TIGIT, and downregulation of the immune activation marker, dipeptidylpeptidase, DPP4. Treatment-associated declines in serum PSA were correlated with induction of Th1 response. In contrast, rises in serum PSA while on treatment were associated with the induction of multiple immune checkpoints, including CTLA4, CEACAM6 and TIGIT. This could represent adaptive immune resistance mechanisms induced by treatment. Taken together, neoadjuvant sipuleucel-T can induce both a Th1 response and negative immune regulation in the prostate cancer microenvironment.

Traumatic optic neuropathy (TON) is an acute injury of the optic nerve secondary to trauma. Loss of retinal ganglion cells (RGCs) is a key pathological process in TON, yet mechanisms responsible for RGC death remain unclear. In a mouse model of TON, real-time noninvasive imaging revealed a dramatic increase in leukocyte rolling and adhesion in veins near the optic nerve (ON) head at 9 hours after ON injury. Although RGC dysfunction and loss were not detected at 24 hours after injury, massive leukocyte infiltration was observed in the superficial retina. These cells were identified as T cells, microglia/monocytes, and neutrophils but not B cells. CXCL10 is a chemokine that recruits leukocytes after binding to its receptor C-X-C chemokine receptor (CXCR) 3. The levels of CXCL10 and CXCR3 were markedly elevated in TON, and up-regulation of CXCL10 was mediated by STAT1/3. Deleting CXCR3 in leukocytes significantly reduced leukocyte recruitment, and prevented RGC death at 7 days after ON injury. Treatment with CXCR3 antagonist attenuated TON-induced RGC dysfunction and cell loss. In vitro co-culture of primary RGCs with leukocytes resulted in increased RGC apoptosis, which was exaggerated in the presence of CXCL10. These results indicate that leukocyte recruitment in retinal vessels near the ON head is an early event in TON and the CXCL10/CXCR3 axis has a critical role in recruiting leukocytes and inducing RGC death.