Optimization of a mouse model of pancreatic cancer to simulate the human phenotypes of metastasis and cachexia.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) presents with a high mortality rate. Two important features of PDAC contribute to this poor outcome. The first is metastasis which occurs in ~ 80% of PDAC patients. The second is cachexia, which compromises treatment tolerance for patients and reduces their quality of life. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging. METHODS: Here, we optimize an orthotopic mouse model of PDAC by altering several conditions, including the subcloning of parental murine PDAC cells, implantation site, number of transplanted cells, and age of recipient mice. We perform spatial profiling to compare primary and metastatic immune microenvironments and RNA sequencing to gain insight into the mechanisms of muscle wasting in PDAC-induced cachexia, comparing non-metastatic to metastatic conditions. RESULTS: These modifications extend the time course of the disease and concurrently increase the rate of metastasis to approximately 70%. Furthermore, reliable cachexia endpoints are achieved in both PDAC mice with and without metastases, which is reminiscent of patients. We also find that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis. CONCLUSION: Together, this model is likely to be advantageous in both advancing our understanding of the mechanism of PDAC cachexia, as well as in the evaluation of novel therapeutics.

Osteosarcoma tumors maintain intra-tumoral transcriptional heterogeneity during bone and lung colonization.

BACKGROUND: Tumors are complex tissues containing collections of phenotypically diverse malignant and nonmalignant cells. We know little of the mechanisms that govern heterogeneity of tumor cells nor of the role heterogeneity plays in overcoming stresses, such as adaptation to different microenvironments. Osteosarcoma is an ideal model for studying these mechanisms-it exhibits widespread inter- and intra-tumoral heterogeneity, predictable patterns of metastasis, and a lack of clear targetable driver mutations. Understanding the processes that facilitate adaptation to primary and metastatic microenvironments could inform the development of therapeutic targeting strategies. RESULTS: We investigated single-cell RNA-sequencing profiles of 47,977 cells obtained from cell line and patient-derived xenograft models as cells adapted to growth within primary bone and metastatic lung environments. Tumor cells maintained phenotypic heterogeneity as they responded to the selective pressures imposed during bone and lung colonization. Heterogenous subsets of cells defined by distinct transcriptional profiles were maintained within bone- and lung-colonizing tumors, despite high-level selection. One prominent heterogenous feature involving glucose metabolism was clearly validated using immunofluorescence staining. Finally, using concurrent lineage tracing and single-cell transcriptomics, we found that lung colonization enriches for multiple clones with distinct transcriptional profiles that are preserved across cellular generations. CONCLUSIONS: Response to environmental stressors occurs through complex and dynamic phenotypic adaptations. Heterogeneity is maintained, even in conditions that enforce clonal selection. These findings likely reflect the influences of developmental processes promoting diversification of tumor cell subpopulations, which are retained, even in the face of selective pressures.

Discharge with Home Health Care is Associated with Increased Complications, Readmission, and Mortality Following Total Shoulder Arthroplasty.

INTRODUCTION: Patients undergoing primary anatomic and reverse total shoulder arthroplasty (TSA) are often discharged with home health care (HHC) to provide access to at-home services and facilitate postoperative recovery and continued medical management. The purpose of this study is to evaluate the short-term postoperative outcomes of patients following primary TSA discharged with HHC, including medical and surgical complications, total cost of care, and total hospital length of stay (LOS). METHODS: The Nationwide Readmissions Database (NRD) was reviewed for patients who underwent elective primary TSA between 2016 to 2020 for a retrospective cohort analysis. Patients were stratified by discharge status following the inpatient admission, with 32,497 patients discharged with HHC and 116,402 patients discharged routinely with self-care. Patient demographics, preoperative medical comorbidities, postoperative medical and surgical complications within 180 days, cost of admission, and total hospital length of stay (LOS) were compared between the two discharge groups using Chi-squared analyses. Further multivariate analysis was conducted to control for independent prognosticators on the effect of HHC on postoperative outcomes. RESULTS: Discharge with HHC was correlated with significantly increased rates of all-cause medical complications (OR 1.6, p < 0.001), surgical site infection (SSI) (OR 2.8, p < 0.001), hospital readmission (OR 1.3, p < 0.001), and death (OR 2.1, p < 0.001) within 180 days of primary TSA. Multivariate analysis suggests these correlations are independent risk factors and not due to patient demographics or preoperative medical comorbidities. While discharge with HHC was found to be associated with increased hospital LOS (1.8 vs. 1.3 days, p < 0.001), there were no significant observed differences in cost of care. CONCLUSION: This study demonstrates that discharge with HHC compared to routine discharge while accounting for several preoperative comorbidities and demographic variables is associated with increased medical complications, SSI, readmission, and death within 180 days of TSA, but no increase in overall patient cost. These findings suggest HHC disposition status can serve as a prognosticator for postoperative complications and can help guide clinician decision making when determining appropriate surgical candidacy.

Muscle inflammation is regulated by NF-κB from multiple cells to control distinct states of wasting in cancer cachexia.

Although cancer cachexia is classically characterized as a systemic inflammatory disorder, emerging evidence indicates that weight loss also associates with local tissue inflammation. We queried the regulation of this inflammation and its causality to cachexia by exploring skeletal muscle, whose atrophy strongly associates with poor outcomes. Using multiple mouse models and patient samples, we show that cachectic muscle is marked by enhanced innate immunity. Nuclear factor κB (NF-κB) activity in multiple cells, including satellite cells, myofibers, and fibro-adipogenic progenitors, promotes macrophage expansion equally derived from infiltrating monocytes and resident cells. Moreover, NF-κB-activated cells and macrophages undergo crosstalk; NF-κB+ cells recruit macrophages to inhibit regeneration and promote atrophy but, interestingly, also protect myofibers, while macrophages stimulate NF-κB+ cells to sustain an inflammatory feedforward loop. Together, we propose that NF-κB functions in multiple cells in the muscle microenvironment to stimulate macrophages that both promote and protect against muscle wasting in cancer.

Noncanonical activation of GLI signaling in SOX2+ cells drives medulloblastoma relapse.

SRY (sex determining region Y)-box 2 (SOX2)-labeled cells play key roles in chemoresistance and tumor relapse; thus, it is critical to elucidate the mechanisms propagating them. Single-cell transcriptomic analyses of the most common malignant pediatric brain tumor, medulloblastoma (MB), revealed the existence of astrocytic Sox2+ cells expressing sonic hedgehog (SHH) signaling biomarkers. Treatment with vismodegib, an SHH inhibitor that acts on Smoothened (Smo), led to increases in astrocyte-like Sox2+ cells. Using SOX2-enriched MB cultures, we observed that SOX2+ cells required SHH signaling to propagate, and unlike in the proliferative tumor bulk, the SHH pathway was activated in these cells downstream of Smo in an MYC-dependent manner. Functionally different GLI inhibitors depleted vismodegib-resistant SOX2+ cells from MB tissues, reduced their ability to further engraft in vivo, and increased symptom-free survival. Our results emphasize the promise of therapies targeting GLI to deplete SOX2+ cells and provide stable tumor remission.