Chordoma cancer stem cell subpopulation characterization may guide targeted immunotherapy approaches to reduce disease recurrence.

Introduction: Cancer stem cells (CSCs), a group of tumor-initiating and tumor-maintaining cells, may be major players in the treatment resistance and recurrence distinctive of chordoma. Characterizing CSCs is crucial to better targeting this subpopulation. Methods: Using flow cytometry, six chordoma cell lines were evaluated for CSC composition. In vitro, cell lines were stained for B7H6, HER2, MICA-B, ULBP1, EGFR, and PD-L1 surface markers. Eighteen resected chordomas were stained using a multispectral immunofluorescence (mIF) antibody panel to identify CSCs in vivo. HALO software was used for quantitative CSC density and spatial analysis. Results: In vitro, chordoma CSCs express more B7H6, MICA-B, and ULBP1, assessed by percent positivity and mean fluorescence intensity (MFI), as compared to non-CSCs in all cell lines. PD- L1 percent positivity is increased by >20% in CSCs compared to non-CSCs in all cell lines except CH22. In vivo, CSCs comprise 1.39% of chordoma cells and most are PD-L1+ (75.18%). A spatial analysis suggests that chordoma CSCs cluster at an average distance of 71.51 mm (SD 73.40 mm) from stroma. Discussion: To our knowledge, this study is the first to identify individual chordoma CSCs and describe their surface phenotypes using in vitro and in vivo methods. PD-L1 is overexpressed on CSCs in chordoma human cell lines and operative tumor samples. Similarly, potential immunotherapeutic targets on CSCs, including B7H6, MICA-B, ULBP1, EGFR, and HER2 are overexpressed across cell lines. Targeting these markers may have a preferential role in combating CSCs, an aggressive subpopulation likely consequential to chordoma's high recurrence rate.

Multi-spectral immunofluorescence evaluation of the myeloid, T cell, and natural killer cell tumor immune microenvironment in chordoma may guide immunotherapeutic strategies.

Background: Chordoma is a rare, invasive, and devastating bone malignancy of residual notochord tissue that arises at the skull base, sacrum, or spine. In order to maximize immunotherapeutic approaches as a potential treatment strategy in chordoma it is important to fully characterize the tumor immune microenvironment (TIME). Multispectral immunofluorescence (MIF) allows for comprehensive evaluation of tumor compartments, molecular co-expression, and immune cell spatial relationships. Here we implement MIF to define the myeloid, T cell, and natural killer (NK) cell compartments in an effort to guide rational design of immunotherapeutic strategies for chordoma. Methods: Chordoma tumor tissue from 57 patients was evaluated using MIF. Three panels were validated to assess myeloid cell, T cell, and NK cell populations. Slides were stained using an automated system and HALO software objective analysis was utilized for quantitative immune cell density and spatial comparisons between tumor and stroma compartments. Results: Chordoma TIME analysis revealed macrophage infiltration of the tumor parenchyma at a significantly higher density than stroma. In contrast, helper T cells, cytotoxic T cells, and T regulatory cells were significantly more abundant in stroma versus tumor. T cell compartment infiltration more commonly demonstrated a tumor parenchymal exclusion pattern, most markedly among cytotoxic T cells. NK cells were sparsely found within the chordoma TIME and few were in an activated state. No immune composition differences were seen in chordomas originating from diverse anatomic sites or between those resected at primary versus advanced disease stage. Conclusion: This is the first comprehensive evaluation of the chordoma TIME including myeloid, T cell, and NK cell appraisal using MIF. Our findings demonstrate that myeloid cells significantly infiltrate chordoma tumor parenchyma while T cells tend to be tumor parenchymal excluded with high stromal infiltration. On average, myeloid cells are found nearer to target tumor cells than T cells, potentially resulting in restriction of T effector cell function. This study suggests that future immunotherapy combinations for chordoma should be aimed at decreasing myeloid cell suppressive function while enhancing cytotoxic T cell and NK cell killing.

Dynamics and Outcomes of Plasmodium Infections in Grammomys surdaster (Grammomys dolichurus) Thicket Rats versus Inbred Mice.

Investigations of malaria infection are often conducted by studying rodent Plasmodium species in inbred laboratory mice, but the efficacy of vaccines or adjunctive therapies observed in these models often does not translate to protection in humans. This raises concerns that mouse malaria models do not recapitulate important features of human malaria infections. African woodland thicket rats (Grammomys surdaster) are the natural host for the rodent malaria parasite Plasmodium berghei and the suspected natural host for Plasmodium vinckei vinckei. Previously, we reported that thicket rats are highly susceptible to diverse rodent parasite species, including P. berghei, Plasmodium yoelii, and Plasmodium chabaudi chabaudi, and are a more stringent model to assess the efficacy of whole-sporozoite vaccines than laboratory mice. Here, we compare the course of infection and virulence with additional rodent Plasmodium species, including various strains of P. berghei, P. yoelii, P. chabaudi, and P. vinckei, in thicket rats versus laboratory mice. We present evidence that rodent malaria parasite growth typically differs between the natural versus nonnatural host; G. surdaster limit infection by multiple rodent malaria strains, delaying and reducing peak parasitemia compared with laboratory mice. The course of malaria infection in thicket rats varied depending on parasite species and strain, resulting in self-cure, chronic parasitemia, or rapidly lethal infection, thus offering a variety of rodent malaria models to study different clinical outcomes in the natural host.

Grammomys surdaster, the Natural Host for Plasmodium berghei Parasites, as a Model to Study Whole-Organism Vaccines Against Malaria.

AbstractInbred mice are commonly used to test candidate malaria vaccines, but have been unreliable for predicting efficacy in humans. To establish a more rigorous animal model, we acquired African woodland thicket rats of the genus Grammomys, the natural hosts for Plasmodium berghei. Thicket rats were acquired and identified as Grammomys surdaster by skull and teeth measurements and mitochondrial DNA genotyping. Herein, we demonstrate that thicket rats are highly susceptible to infection by P. berghei, and moderately susceptible to Plasmodium yoelii and Plasmodium chabaudi: 1-2 infected mosquito bites or 25-100 sporozoites administered by intravenous injection consistently resulted in patent parasitemia with P. berghei, and resulted in patent parasitemia with P. yoelii and P. chabaudi strains for at least 50% of animals. We then assessed efficacy of whole-organism vaccines to induce sterile immunity, and compared the thicket rat model to conventional mouse models. Using P. berghei ANKA radiation-attenuated sporozoites, and P. berghei ANKA and P. yoelii chemoprophylaxis vaccination approaches, we found that standard doses of vaccine sufficient to protect laboratory mice for a long duration against malaria challenge, are insufficient to protect thicket rats, which require higher doses of vaccine to achieve even short-term sterile immunity. Thicket rats may offer a more stringent and pertinent model for evaluating whole-organism vaccines.