Lung Cancer Pathological Image Analysis Using a Hidden Potts Model

Pediatric high-grade gliomas (PHGG) are aggressive brain tumors with 5-year survival rates ranging from <2% to 20% depending upon subtype. PHGG presents differently from patient to patient and is intratumorally heterogeneous, posing challenges in designing therapies. We hypothesized that heterogeneity occurs because PHGG comprises multiple distinct tumor and immune cell types in varying proportions, each of which may influence tumor characteristics. We obtained 19 PHGG samples from our institution's pediatric brain tumor bank. We constructed a comprehensive transcriptomic dataset at the single-cell level using single-cell RNA-Seq (scRNA-Seq), identified known glial and immune cell types, and performed differential gene expression and gene set enrichment analysis. We conducted multi-channel immunofluorescence (IF) staining to confirm the transcriptomic results. Our PHGG samples included 3 principal predicted tumor cell types: astrocytes, oligodendrocyte progenitors (OPCs), and mesenchymal-like cells (Mes). These cell types differed in their gene expression profiles, pathway enrichment, and mesenchymal character. We identified a macrophage population enriched in mesenchymal and inflammatory gene expression as a possible source of mesenchymal tumor characteristics. We found evidence of T-cell exhaustion and suppression. PHGG comprises multiple distinct proliferating tumor cell types. Microglia-derived macrophages may drive mesenchymal gene expression in PHGG. The predicted Mes tumor cell population likely derives from OPCs. The variable tumor cell populations rely on different oncogenic pathways and are thus likely to vary in their responses to therapy.

&lt;div&gt;Abstract&lt;p&gt;The spatial organization of different types of cells in tumor tissues reveals important information about the tumor microenvironment (TME). To facilitate the study of cellular spatial organization and interactions, we developed Histology-based Digital-Staining, a deep learning-based computation model, to segment the nuclei of tumor, stroma, lymphocyte, macrophage, karyorrhexis, and red blood cells from standard hematoxylin and eosin–stained pathology images in lung adenocarcinoma. Using this tool, we identified and classified cell nuclei and extracted 48 cell spatial organization-related features that characterize the TME. Using these features, we developed a prognostic model from the National Lung Screening Trial dataset, and independently validated the model in The Cancer Genome Atlas lung adenocarcinoma dataset, in which the predicted high-risk group showed significantly worse survival than the low-risk group (&lt;i&gt;P&lt;/i&gt; = 0.001), with a HR of 2.23 (1.37–3.65) after adjusting for clinical variables. Furthermore, the image-derived TME features significantly correlated with the gene expression of biological pathways. For example, transcriptional activation of both the T-cell receptor and programmed cell death protein 1 pathways positively correlated with the density of detected lymphocytes in tumor tissues, while expression of the extracellular matrix organization pathway positively correlated with the density of stromal cells. In summary, we demonstrate that the spatial organization of different cell types is predictive of patient survival and associated with the gene expression of biological pathways.&lt;/p&gt;Significance:&lt;p&gt;These findings present a deep learning-based analysis tool to study the TME in pathology images and demonstrate that the cell spatial organization is predictive of patient survival and is associated with gene expression.&lt;/p&gt;&lt;p&gt;&lt;i&gt;See related commentary by Rodriguez-Antolin, p. 1912&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

Expression and Function of β1 Integrins on Adherent and Nonadherent Ehrlich Ascites Tumor Cells

Tumor masses contain a diverse array of cell types, including both normal and tumor cells from different lineages, endothelial cells, and immune cells. Interactions among these cellular constituents and their spatial arrangements play a pivotal role in tumor development, progression, and metastasis. While single-cell analysis has provided valuable insights regarding the dissociated cellular composition of tumors, it falls short in elucidating the complex interplay of cell-cell interactions and spatial relationships that may drive tumor cellular behavior and gene expression. We used the Curio Seeker Spatial Mapping Kits to uncover those spatial relationships in human tumor samples. The method captured whole transcriptomes and preserved the spatial locations of tumor cells, infiltrating immune cells, and normal cells by self segregating gene expression signatures and unbiased clustering. Amongst other findings by using Curio Seeker, we identified areas of immune cell infiltrates that were devoid of tumor cells, signifying zones of potential anti-tumor response. The high spatial resolution, continuous gene expression maps from the Curio Seeker Spatial Mapping Kits reveal spatial interactions between different cellular populations that enable researchers to better understand how the interplay between tumor cell types and their cellular neighborhoods affect gene expression and tumor cell behavior. Citation Format: Marie M. Lee, Julie Wilhelmy, Thomas Ong, Wanxin Wang, Bertrand Yeung, Christina Chang, Christina Fan. Investigating unbiased whole transcriptome high-resolution spatial interactions within tumor masses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3796.

How standard care shapes the cellular composition of recurrent medulloblastoma (MB), if therapy selects for specific tumor or immune cell types, is unknown. We report the pilot phase of our ongoing effort to profile human longitudinal MB specimens via single-cell transcriptomics and epigenetics. We profiled 11 diagnostic and eight recurrent specimens from 19 subjects via single-nucleus RNA sequencing (snRNA-seq), and four subjects via single-nucleus assay for transposase-accessible chromatin. Specimens from select subjects were also profiled to assess genome-wide enhancer activity via single-nucleus cleavage-under-targets and tagmentation. We found an upregulation of the DNA-damage response, RNA translation, WNT and NOTCH signaling in recurrent specimens. The percentages of stem-like cells increased by over two-fold at recurrence. We found that microglia and oligodendrocyte-lineage cells were the most abundant non-malignant tumor-associated cell types, representing 2%-10% of cells profiled. Microglia abundances were relatively stable across molecular subtypes, and when comparing primary to recurrent tumors. There was a moderate, but statistically significant, increase in oligodendrocyte abundance in SSH and WNT tumors, compared to Group 3/4 tumors. We compared gene expression in tumor cells with public snRNA-seq from developing human cerebella (PCW 9-21). Combined Group-3/4 cell analysis supports a common lineage hierarchy, with an enrichment for unipolar brush-cell and Purkinje-cell phenotypes found in Group-4 tumors. All Group-3/4 cases contained cycling cells expressing markers of PAX2+ interneuron progenitors, most cycling cells had this phenotype. All specimens contained populations of non-cycling granule-cell progenitor-like cells. We performed single-cell co-expression receptor/ligand analysis to infer paracrine signaling between tumor and non-malignant cell types. This identified both tumor cells and microglia as sources of growth factors, pro-inflammatory cytokines, and pro-apoptotic ligands. Non-malignant oligodendrocyte-lineage cells uniquely expressed IL6-family cytokines, pleiotrophin, and class-III semaphorins. These studies shed light on the cellular heterogeneity of MB and the effect of standard therapy in shaping composition at recurrence.

This is a study of 269 patients with lung cancer at the Erie County Medical Center who were admitted between 1973 and 1978. They were analyzed for sex, race, age, history of smoking, occupation, tumor cell type, cytology, incidence of metastases, changes in liver function, mode of treatment, and survival. The incidence of cancer was highest in white males. Only 1.5% of patients were under age 40. Smoking was a predisposing factor. Not enough information was available to determine the relationship of occupation to lung cancer. Squamous cell carcinoma was the most common (53.9%), followed by adenocarcinoma (16.0%) and small-cell carcinoma (12.6%). Sputum cytology was 28.3% sensitive, and bronchial washings were 52.2% sensitive. A greater incidence of bone metastases from a small-cell primary (50%) was found than is reported in the literature. Changes in SGOT and/or SGPT liver enzymes correlated significantly with liver metastases, but not with tumor cell type. Changes in alkaline phosphatase correlated well with bone metastases. Radiation was the most commonly used mode of therapy. The best survival was achieved in patients treated by surgery (22.6 months), followed by surgery and radiation (16.2 months); those treated by radiation alone had a mean survival time of 8.7 months. Untreated patients had a mean survival time of 2.4 months. Treated patients with adenocarcinomas had the longest survival (18.5 months), compared to 13.0 months for those with squamous cell carcinomas and 8.4 months for those with small-cell carcinomas. Only three patients survived 5 years, all of whom were treated surgically for adenocarcinoma. No patients with tumors of other cell types survived 5 years. The 5-year survival rates are 2.1% (3 of 141) for all histologic types of lung cancer and 12.5% (3 of 24) for adenocarcinoma.

Beta 2-Microglobulin (beta 2-M) was analysed in serum of 145 patients with renal cell carcinoma, and serum creatinine < 125 mumol/1 by a radioimmunometric method. Forty-nine (34%) patients had serum beta 2-M level > or = 3.0 mg/l. Of the patients with distal metastases 46% had elevated levels, compared with 19% with stage I disease. Serum beta 2-M correlated with histopathologic grade; 58% of the patients with poorly differentiated (grade 4) tumours had elevated levels compared with 18% in grade 1-2 tumours. Also tumour cell type was associated with serum beta 2-M; 52% of the patients with plasmic tumours had elevated levels compared with 6% in the clear cell type. In a univariate prognostic analysis elevated serum beta 2-M level was inversely correlated with survival time. Using a multivariate analysis the strong prognostic factors were clinical stage and tumour diameter. Weaker factors were age and cell type, whereas the prognostic value of serum beta 2-M disappeared. However, if tumour cell type was excluded from the analysis, serum beta 2-M was identified as a prognostic factor.

Highly sensitive monitoring of telomerase activity in living cells based on rapidly triggered cascade amplification reaction

Glutamine addiction of cancer cells is thought to be an attractive way to prevent cancer growth and spreading. Although some regulatory factors of glutamine consumption are known, the actual cell-type specific regulatory mechanism hasn’t been explored yet. In breast tumors, there are two types of tumor cells that may be malignantly transformed from different cellular origins with dramatically different gene expression. We found distinct glutamine requirement in these two types of cells: luminal epithelial cells, as well as their corresponding transformed tumor cells, are more independent to extracellular glutamine than basal cells. This luminal- specific glutamine independent is regulated by its specific expression of glutamine synthetase, which can produce glutamine by combing glutamate and amine group. In response to glutamine deprivation, the expression of glutamine synthetase in luminal cells allow them to produce and export glutamine to the extracellular space to support the survival of glutamine addicted basal cells in a mechanism of metabolic symbiosis. By these observations, the glutamine deprivation therapy should be performed more efficiently with the help of glutamine synthetase inhibition.

The cytology of 6 osteosarcomas was examined by electron microscopy. In keeping with the varied pattern of osteosarcomas seen by light microscopy several types of tumor cells could be differentiated: osteoblast-like, fibroblast-like, chondroblast-like, osteoclast-like and histiocyte-like cells. Moreover, atypical malignant mesenchymal cells and vascular spaces were present. The individual cytoplasmic organelles are not considered to be specific to particular types of cell as seen from the discussion of the significance of rough endoplasmic reticulum, microfilaments and lysosomes. Only examination of the composite pattern of subcellular organelles allows the differentiation of certain cell types. All tumor cells visible in osteosarcomas are considered as modifications of a transformed common progenitor cell. Because of the variegated cytological picture a multipotent mesenchymal cell rather than an osteoblastic cell is assumed to be the ancestor cell.

Xp11.2 translocation renal neoplasm with features of TFE3 rearrangement associated renal cell carcinoma and Xp11 translocation renal mesenchymal tumor with melanocytic differentiation harboring NONO-TFE3 fusion gene

Tumor cells are malignant derivatives of normal cells. There are characteristic differences in the mechanophysical properties of normal and tumor cells, and these differences stem from the changes that occur in the cell cytoskeleton during cancer progression. There is a need for viable whole blood processing techniques for rapid and reliable tumor cell detection that do not require tagging. Micropore biosensors have previously been used to differentiate tumor cells from normal cells and we have used a micropore-based electromechanical transducer to differentiate one type of tumor cells from the other types. This device generated electrical signals that were characteristic of the cell properties. Three non-small cell lung cancer (NSCLC) cell lines, NCl-H1155, A549 and NCI-H460, were successfully differentiated. NCI-H1155, due to their comparatively smaller size, were found to be the quickest in translocating through the micropore. Their translocation through a 15 μm micropore caused electrical pulses with an average translocation time of 101 ± 9.4 μs and an average peak amplitude of 3.71 ± 0.42 μA, whereas translocation of A549 and NCI-H460 caused pulses with average translocation times of 126 ± 17.9 μs and 148 ± 13.7 μs and average peak amplitudes of 4.58 ± 0.61 μA and 5.27 ± 0.66 μA, respectively. This transformation of the differences in cell properties into differences in the electrical profiles (i.e. the differences in peak amplitudes and translocation times) with this electromechanical transducer is a quantitative way to differentiate these lung cancer cells. The solid-state micropore device processed whole biological samples without any pre-processing requirements and is thus ideal for point-of-care applications.

Tumor cell type can be reproducibly diagnosed and is of independent prognostic significance in patients with maximally debulked ovarian carcinoma

Mechanical forces from non-ablative pulsed focused ultrasound (pFUS) generate pro-inflammatory tumor microenvironments (TME), marked by increased cytokines, chemokines, and trophic factors, as well as immune cell infiltration and reduced tumor growth. pFUS also causes DNA damage within tumors, which is a potent activator of immunity and could contribute to changes in the TME. This study investigated mechanisms behind the mechanotransductive effects of pFUS causing DNA damage in several tumor cell types.Methods: 4T1 (murine breast tumor), B16 (murine melanoma), C6 (rat glioma), or MDA-MB-231 (human breast tumor) cells were sonicated in vitro (1.1MHz; 6MPa PNP; 10ms pulses; 10% duty cycle; 300 pulses). DNA damage was detected by TUNEL, apoptosis was measured by immunocytochemistry for cleaved caspase-3. Calcium, superoxide, and H2O2 were detected by fluorescent indicators and modulated by BAPTA-AM, mtTEMPOL, or Trolox, respectively.Results: pFUS increased TUNEL reactivity (range = 1.6-2.7-fold) in all cell types except C6 and did not induce apoptosis in any cell line. All lines displayed cytosolic Ca2+ transients during sonication. pFUS increased superoxide (range = 1.6-2.0-fold) and H2O2 (range = 2.3-2.8-fold) in all cell types except C6. BAPTA-AM blocked increased TUNEL reactivity, superoxide and H2O2 formation, while Trolox also blocked increased TUNEL reactivity increased after pFUS. mtTEMPOL allowed H2O2 formation and did not block increased TUNEL reactivity after pFUS. Unsonicated C6 cells had higher baseline concentrations of cytosolic Ca2+, superoxide, and H2O2, which were not associated with greater baseline TUNEL reactivity than the other cell lines.Conclusions: Mechanotransduction of pFUS directly induces DNA damage in tumor cells by cytosolic Ca2+ transients causing formation of superoxide and subsequently, H2O2. These results further suggest potential clinical utility for pFUS. However, the lack of pFUS-induced DNA damage in C6 cells demonstrates a range of potential tumor responses that may arise from physiological differences such as Ca2+ or redox homeostasis.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.