Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in > 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell–cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC. Significance: A high-throughput pipeline using multiplex immunofluorescence in pancreatic cancer reveals striking expression subtype intratumoral heterogeneity with implications for therapy selection and identifies co-expressor cells that may serve as intermediates during subtype switching.

Purpose: Neoadjuvant chemotherapy is increasingly administered to patients with resectable or borderline resectable pancreatic ductal adenocarcinoma (PDAC), yet its impact on the tumor immune microenvironment is incompletely understood. Experimental: Design: We employed quantitative, spatially resolved multiplex immunofluorescence and digital image analysis to identify T-cell subpopulations, macrophage polarization states, and myeloid cell subpopulations in a multi-institution cohort of up-front resected primary tumors (n = 299) and in a comparative set of resected tumors after FOLFIRINOX-based neoadjuvant therapy (n = 36) or up-front surgery (n = 30). Multivariable-adjusted Cox proportional hazards models were used to evaluate associations between the immune microenvironment and patient outcomes. Results: In the multi-institutional resection cohort, immune cells exhibited substantial heterogeneity across patient tumors and were located predominantly in stromal regions. Unsupervised clustering using immune cell densities identified four main patterns of immune cell infiltration. One pattern, seen in 20% of tumors and characterized by abundant T cells (T cell–rich) and a paucity of immunosuppressive granulocytes and macrophages, was associated with improved patient survival. Neoadjuvant chemotherapy was associated with a higher CD8:CD4 ratio, greater M1:M2–polarized macrophage ratio, and reduced CD15+ARG1+ immunosuppressive granulocyte density. Within neoadjuvant-treated tumors, 72% showed a T cell–rich pattern with low immunosuppressive granulocytes and macrophages. M1-polarized macrophages were located closer to tumor cells after neoadjuvant chemotherapy, and colocalization of M1-polarized macrophages and tumor cells was associated with greater tumor pathologic response and improved patient survival. Conclusions: Neoadjuvant chemotherapy with FOLFIRINOX shifts the PDAC immune microenvironment toward an anti-tumorigenic state associated with improved patient survival.

This study evaluated the effect of time of botulinum toxin A (BoNT-A) treatment on clinical outcomes in adults with post-stroke spasticity (PSS). Individual data were pooled from five studies. Eligible patients received ≥1 BoNT-A injection(s) for PSS and had goal attainment scaling (GAS) scores measured at baseline and 12 weeks. Patients were grouped according to time of treatment post-stroke: early (<1 year) or late (≥1 year). The primary endpoint was the total GAS (GAS-T) score change from baseline to 12 weeks. Secondary outcomes included the proportion of patients with a GAS-T score ≥ 50. Overall, 968 patients were included (166 early and 802 late). Median time post-stroke to BoNT-A treatment was 0.5 (early) versus 5.4 (late) years. Mean (standard deviation [SD]) baseline GAS scores were similar between cohorts (early: 36.9 [3.5]; late: 36.9 [3.6]). The mean (SD) change in the GAS-T score from baseline to 12 weeks was greater in the early versus late cohort (15.7 [8.9] vs. 13.1 [8.9]; p < 0.001). More patients in the early versus late cohort had a GAS-T score ≥ 50 (63.9% vs. 47.4%; p < 0.001) at 12 weeks. No new safety concerns were reported. Early treatment of PSS with BoNT-A has a positive impact on patients’ ability to achieve treatment goals. Plain Language Summary: After a stroke, people can experience muscle stiffness in their limbs, called post-stroke spasticity (PSS), which can lead to pain and make movement difficult. Treatment can include botulinum toxin A (BoNT-A) injections given directly into affected muscles. The aim of our study was to assess whether giving BoNT-A within a year after experiencing a stroke was more effective in treating PSS than delaying treatment. We combined data from 968 patients across five different studies. Most people (802 patients) received BoNT-A treatment 1 year or more after their stroke (late treatment group), while 166 people received treatment within a year of their stroke (early treatment group). In the studies, patients set treatment goals with their physician, for example being able to hold an object or walk a certain distance. After treatment, the extent to which each goal was achieved was assessed and scored based on whether the result was less than expected, as expected, or better than expected by the patient and physician. The scores from the two treatment groups were compared. People in the early treatment group did better in achieving their treatment goals compared with those in the late treatment group. We also looked at any side effects patients experienced. No unexpected side effects were reported. BoNT-A treatment of PSS can help patients achieve their treatment goals, and patients treated early (within 1 year after stroke) may do better than those treated later. This information may help in rehabilitation planning for stroke patients.

Although vitamin D (VitD) exhibits antitumor activity in colorectal cancer preclinically, its effects in the human tumor microenvironment (TME) remain unclear. We conducted a randomized, placebo-controlled trial of preoperative high-dose VitD supplementation in patients with stage I to III colon cancer to assess its impact on the TME. Forty-two patients received either VitD3 (50,000 IU/day for 7 days, then 10,000 IU/day) or placebo before surgery. Spatial immune profiling and assessment of VitD receptor (VDR) and CYP27B1 expression were performed on paired tumor samples from 24 patients. VitD significantly increased plasma 25-hydroxyvitamin D levels (P < 0.001), increased CD3+CD8+ memory T cells (P = 0.03), reduced CD3+CD4+FoxP3+ regulatory T cells (P = 0.02), and spatially re-organized the TME, leading to greater T-cell and tumor cell proximity. Posttreatment VDR expression was heterogeneous and decreased overall (P = 0.02). Spatial transcriptomic profiling of posttreatment resections reflected predominantly repressive VDR activity. These findings support an immunomodulatory role for VitD, warranting further mechanistic investigation. Significance: Colorectal cancer remains a leading cause of cancer-related death with limited long-term survival. We evaluated the effects of short-term, preoperative high-dose VitD supplementation on the colon cancer microenvironment in a randomized, placebo-controlled trial. Our findings support VitD as an immune modulator and may inform future treatment strategies.

Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in > 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell–cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC.Significance:A high-throughput pipeline using multiplex immunofluorescence in pancreatic cancer reveals striking expression subtype intratumoral heterogeneity with implications for therapy selection and identifies co-expressor cells that may serve as intermediates during subtype switching.