Losartan controls immune checkpoint blocker-induced edema and improves survival in glioblastoma mouse models.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and CD8+ T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following antiprogrammed death 1 (PD1) antibody treatment that disrupts the blood-tumor barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.

Dendritic cell paucity in mismatch repair-proficient colorectal cancer liver metastases limits immune checkpoint blockade efficacy.

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC.

What's driving girls' adolescent marriage decline in Rakai, Uganda? The interplay between adolescent pregnancies and social determinants, 1999 - 2018.

Few studies have sought to untangle the influence of social determinants and pregnancy on adolescent marriage declines. Using longitudinal data from 15- to 17-year-old girls in the Rakai Community Cohort Survey, we assessed how education, socio-economic status, orphanhood and pregnancy contributed to trends in adolescent marriage. We examined descriptive trends and logistic regressions of the associations between social determinants and adolescent marriage, and conducted causal mediation analysis to assess the extent that pregnancy mediated the effect of education on marriage. Between 1999-2018, adolescent marriages and pregnancies dropped substantially (24%-6% and 28%-8%). Girls' secondary schooling was strongly associated with lower marriage risk (aOR marriage=0.09; 95%CI=0.07-0.12), accounting for time. Lower pregnancy rates partially explained the effect of secondary schooling on lower adolescent marriage (aOR indirect effect=0.55; 95%CI=0.421-0.721). Findings affirm the importance of education in preventing adolescent marriages but call attention to the role of pregnancies in influencing adolescent marriages.

Capacity to Consent to Research Among Adolescent-Parent Dyads in Rakai, Uganda.

OBJECTIVES: To assess the cognitive capacity of early, middle, and late adolescents and their parents or guardians to provide informed consent to a population-based cohort study. STUDY DESIGN: Adolescent-parent/guardian dyads including 40 early (n = 80; 10-14 years), 20 middle (15-17 years), and 20 late (18-19 years) adolescents were recruited from the Rakai Community Cohort Study, an open demographic cohort in Uganda. Participants were administered the MacArthur Competence Assessment Tool for Clinical Research, a structured open-ended assessment; interviews were recorded and transcribed. Twenty transcripts were scored independently by two coders; the intraclass correlation coefficient was 0.89. The remaining interviews were scored individually. We compared mean scores for early and middle/late adolescents using a one-sided t test and score differences between parent/guardian and adolescent dyads using two-sided paired t tests. RESULTS: Early adolescents (mean score, 28.8; 95% CI, 27.1-30.5) scored significantly lower (P < .01) than middle/late adolescents (32.4; 31.6-33.1). In paired dyad comparisons, we observed no statistically significant difference in scores between parents/guardians and middle/late adolescents (difference, -0.2; 95% CI, -1.0-0.6). We found a statistically significant difference in scores between parents/guardians and early adolescents (difference, 3.0; 95% CI, 1.2-4.8). CONCLUSIONS: The capacity of adolescents-of different ages and in diverse settings-to comprehend risks, benefits, and other elements of informed consent is a critical but understudied area in research ethics. Our findings support the practice of having middle and late adolescents provide independent informed consent for sexual and reproductive health studies. Early adolescents may benefit from supported decision-making approaches.

A bilateral tumor model identifies transcriptional programs associated with patient response to immune checkpoint blockade.

Immune checkpoint blockade (ICB) is efficacious in many diverse cancer types, but not all patients respond. It is important to understand the mechanisms driving resistance to these treatments and to identify predictive biomarkers of response to provide best treatment options for all patients. Here we introduce a resection and response-assessment approach for studying the tumor microenvironment before or shortly after treatment initiation to identify predictive biomarkers differentiating responders from nonresponders. Our approach builds on a bilateral tumor implantation technique in a murine metastatic breast cancer model (E0771) coupled with anti-PD-1 therapy. Using our model, we show that tumors from mice responding to ICB therapy had significantly higher CD8+ T cells and fewer Gr1+CD11b+ myeloid-derived suppressor cells (MDSCs) at early time points following therapy initiation. RNA sequencing on the intratumoral CD8+ T cells identified the presence of T cell exhaustion pathways in nonresponding tumors and T cell activation in responding tumors. Strikingly, we showed that our derived response and resistance signatures significantly segregate patients by survival and associate with patient response to ICB. Furthermore, we identified decreased expression of CXCR3 in nonresponding mice and showed that tumors grown in Cxcr3-/- mice had an elevated resistance rate to anti-PD-1 treatment. Our findings suggest that the resection and response tumor model can be used to identify response and resistance biomarkers to ICB therapy and guide the use of combination therapy to further boost the antitumor efficacy of ICB.

Placental growth factor promotes tumour desmoplasia and treatment resistance in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC)-a rare liver malignancy with limited therapeutic options-is characterised by aggressive progression, desmoplasia and vascular abnormalities. The aim of this study was to determine the role of placental growth factor (PlGF) in ICC progression. DESIGN: We evaluated the expression of PlGF in specimens from ICC patients and assessed the therapeutic effect of genetic or pharmacologic inhibition of PlGF in orthotopically grafted ICC mouse models. We evaluated the impact of PlGF stimulation or blockade in ICC cells and cancer-associated fibroblasts (CAFs) using in vitro 3-D coculture systems. RESULTS: PlGF levels were elevated in human ICC stromal cells and circulating blood plasma and were associated with disease progression. Single-cell RNA sequencing showed that the major impact of PlGF blockade in mice was enrichment of quiescent CAFs, characterised by high gene transcription levels related to the Akt pathway, glycolysis and hypoxia signalling. PlGF blockade suppressed Akt phosphorylation and myofibroblast activation in ICC-derived CAFs. PlGF blockade also reduced desmoplasia and tissue stiffness, which resulted in reopening of collapsed tumour vessels and improved blood perfusion, while reducing ICC cell invasion. Moreover, PlGF blockade enhanced the efficacy of standard chemotherapy in mice-bearing ICC. Conclusion PlGF blockade leads to a reduction in intratumorous hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in mice-bearing aggressive ICC.

Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking.

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

Reprogramming the microenvironment with tumor-selective angiotensin blockers enhances cancer immunotherapy.

Cancer-associated fibroblasts (CAFs) can either suppress or support T lymphocyte activity, suggesting that CAFs may be reprogrammable to an immunosupportive state. Angiotensin receptor blockers (ARBs) convert myofibroblast CAFs to a quiescent state, but whether ARBs can reprogram CAFs to promote T lymphocyte activity and enhance immunotherapy is unknown. Moreover, ARB doses are limited by systemic adverse effects such as hypotension due to the importance of angiotensin signaling outside tumors. To enhance the efficacy and specificity of ARBs in cancer with the goal of revealing their effects on antitumor immunity, we developed ARB nanoconjugates that preferentially accumulate and act in tumors. We created a diverse library of hundreds of acid-degradable polymers and chemically linked ARBs to the polymer most sensitive to tumor pH. These tumor microenvironment-activated ARBs (TMA-ARBs) remain intact and inactive in circulation while achieving high concentrations in tumors, wherein they break down to active ARBs. This tumor-preferential activity enhances the CAF-reprogramming effects of ARBs while eliminating blood pressure-lowering effects. Notably, TMA-ARBs alleviate immunosuppression and improve T lymphocyte activity, enabling dramatically improved responses to immune-checkpoint blockers in mice with primary as well as metastatic breast cancer.

Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer.

Metastatic breast cancers (mBCs) are largely resistant to immune checkpoint blockade, but the mechanisms remain unclear. Primary breast cancers are characterized by a dense fibrotic stroma, which is considered immunosuppressive in multiple malignancies, but the stromal composition of breast cancer metastases and its role in immunosuppression are largely unknown. Here we show that liver and lung metastases of human breast cancers tend to be highly fibrotic, and unlike primary breast tumors, they exclude cytotoxic T lymphocytes (CTLs). Unbiased analysis of the The Cancer Genome Atlas database of human breast tumors revealed a set of genes that are associated with stromal T-lymphocyte exclusion. Among these, we focused on CXCL12 as a relevant target based on its known roles in immunosuppression in other cancer types. We found that the CXCL12 receptor CXCR4 is highly expressed in both human primary tumors and metastases. To gain insight into the role of the CXCL12/CXCR4 axis, we inhibited CXCR4 signaling pharmacologically and found that plerixafor decreases fibrosis, alleviates solid stress, decompresses blood vessels, increases CTL infiltration, and decreases immunosuppression in murine mBC models. By deleting CXCR4 in αSMA+ cells, we confirmed that these immunosuppressive effects are dependent on CXCR4 signaling in αSMA+ cells, which include cancer-associated fibroblasts as well as other cells such as pericytes. Accordingly, CXCR4 inhibition more than doubles the response to immune checkpoint blockers in mice bearing mBCs. These findings demonstrate that CXCL12/CXCR4-mediated desmoplasia in mBC promotes immunosuppression and is a potential target for overcoming therapeutic resistance to immune checkpoint blockade in mBC patients.

Dual Programmed Death Receptor-1 and Vascular Endothelial Growth Factor Receptor-2 Blockade Promotes Vascular Normalization and Enhances Antitumor Immune Responses in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Activation of the antitumor immune response using programmed death receptor-1 (PD-1) blockade showed benefit only in a fraction of patients with hepatocellular carcinoma (HCC). Combining PD-1 blockade with antiangiogenesis has shown promise in substantially increasing the fraction of patients with HCC who respond to treatment, but the mechanism of this interaction is unknown. APPROACH AND RESULTS: We recapitulated these clinical outcomes using orthotopic-grafted or induced-murine models of HCC. Specific blockade of vascular endothelial receptor 2 (VEGFR-2) using a murine antibody significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 antibody treatment alone conferred a minor survival advantage in one model. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in both models. Combination therapy reprogrammed the immune microenvironment by increasing cluster of differentiation 8-positive (CD8+ ) cytotoxic T cell infiltration and activation, shifting the M1/M2 ratio of tumor-associated macrophages and reducing T regulatory cell (Treg) and chemokine (C-C motif) receptor 2-positive monocyte infiltration in HCC tissue. In these models, VEGFR-2 was selectively expressed in tumor endothelial cells. Using spheroid cultures of HCC tissue, we found that PD-ligand 1 expression in HCC cells was induced in a paracrine manner upon anti-VEGFR-2 blockade in endothelial cells in part through interferon-gamma expression. Moreover, we found that VEGFR-2 blockade increased PD-1 expression in tumor-infiltrating CD4+ cells. We also found that under anti-PD-1 therapy, CD4+ cells promote normalized vessel formation in the face of antiangiogenic therapy with anti-VEGFR-2 antibody. CONCLUSIONS: We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase overall survival in both anti-PD-1 therapy-resistant and anti-PD-1 therapy-responsive HCC models.