Image-based multiplex immune profiling of cancer tissues: translational implications. A report of the International Immuno-oncology Biomarker Working Group on Breast Cancer.

Recent advances in the field of immuno-oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high-throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Spatial analyses of immune cell infiltration in cancer: current methods and future directions: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer.

Modern histologic imaging platforms coupled with machine learning methods have provided new opportunities to map the spatial distribution of immune cells in the tumor microenvironment. However, there exists no standardized method for describing or analyzing spatial immune cell data, and most reported spatial analyses are rudimentary. In this review, we provide an overview of two approaches for reporting and analyzing spatial data (raster versus vector-based). We then provide a compendium of spatial immune cell metrics that have been reported in the literature, summarizing prognostic associations in the context of a variety of cancers. We conclude by discussing two well-described clinical biomarkers, the breast cancer stromal tumor infiltrating lymphocytes score and the colon cancer Immunoscore, and describe investigative opportunities to improve clinical utility of these spatial biomarkers. © 2023 The Pathological Society of Great Britain and Ireland.

Pitfalls in machine learning-based assessment of tumor-infiltrating lymphocytes in breast cancer: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer

The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

1,4-dihydroxy quininib modulates the secretome of uveal melanoma tumour explants and a marker of oxidative phosphorylation in a metastatic xenograft model.

Uveal melanoma (UM) is an intraocular cancer with propensity for liver metastases. The median overall survival (OS) for metastatic UM (MUM) is 1.07 years, with a reported range of 0.84-1.34. In primary UM, high cysteinyl leukotriene receptor 1 (CysLT1) expression associates with poor outcomes. CysLT1 antagonists, quininib and 1,4-dihydroxy quininib, alter cancer hallmarks of primary and metastatic UM cell lines in vitro. Here, the clinical relevance of CysLT receptors and therapeutic potential of quininib analogs is elaborated in UM using preclinical in vivo orthotopic xenograft models and ex vivo patient samples. Immunohistochemical staining of an independent cohort (n = 64) of primary UM patients confirmed high CysLT1 expression significantly associates with death from metastatic disease (p = 0.02; HR 2.28; 95% CI 1.08-4.78), solidifying the disease relevance of CysLT1 in UM. In primary UM samples (n = 11) cultured as ex vivo explants, 1,4-dihydroxy quininib significantly alters the secretion of IL-13, IL-2, and TNF-α. In an orthotopic, cell line-derived xenograft model of MUM, 1,4-dihydroxy quininib administered intraperitoneally at 25 mg/kg significantly decreases ATP5B expression (p = 0.03), a marker of oxidative phosphorylation. In UM, high ATP5F1B is a poor prognostic indicator, whereas low ATP5F1B, in combination with disomy 3, correlates with an absence of metastatic disease in the TCGA-UM dataset. These preclinical data highlight the diagnostic potential of CysLT1 and ATP5F1B in UM, and the therapeutic potential of 1,4-dihydroxy quininib with ATP5F1B as a companion diagnostic to treat MUM.

Deciphering the molecular pathways of apoptosis using purified fractions from leaf extract of Basella alba through studying the regulation of apoptosis related genes.

Apoptosis plays a pivotal role in the exclusion of abnormal cells without any ruin of surrounding healthy cells. Generally, it occurs through an orderly and autonomously process which is controlled by proper function of various genes. Therefore, the current experiments detect the expression level/pattern of those genes to confirm the involvement of extrinsic and intrinsic pathway using Basella alba leaf (BAL). Several fractions after gel filtration chromatography of BAL extract have been pooled to evaluates its apoptosis induction potentiality on Ehrlich's Ascites Carcinoma (EAC) cells through conducting a number of bio-assays such as cell growth inhibition assay, fluorescence and optical microscopy, DNA fragmentation assay and gene expression analysis etc. The pooled fractions of BAL showed 12-56% inhibitory effect on EAC cell line at the concentration range of 25-400 µg/ml that was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. They also exhibited excellent cell growth inhibition at in vivo and in vitro condition when treated with 10, 20 and 40 mg/kg day. After administration of six consequent days, significant morphological features of apoptosis were observed in EAC cells under both fluorescence and optical microscope which was further supported by DNA fragmentation assay. The polymerase chain reaction amplification of bax, bcl-2 (B-cell lymphoma 2), p53, tumor necrosis factor-α, Fas, NF-kß (Nuclear factor-Kappa-B), PARP-1 (Poly (ADP-ribose) polymerase), Cyt-c cas-8, cas-9 and cas-3 revealed that the experimental sample able to induce apoptosis in both extrinsic and intrinsic pathways through altering the gene expression. The current findings suggest that sample from BAL occupy wonderful competence to induce cell apoptosis and become an ideal resource for cancer treatment.

Differential expression of Bax-Bcl-2 and PARP-1 confirms apoptosis of EAC cells in Swiss albino mice by Morus laevigata.

A safer natural alternative to treat neoplastic cells by inducing apoptosis is a prime requisite. Therefore, the current study was to evaluate the antiproliferative activity of Morus laevigata, a wild-type Mulberry species. Antioxidant and cytotoxic activity of aqueous extracts of M. laevigata leaf (MLL) and M. laevigata bark (MLB) were evaluated. The in vivo cell growth inhibition was assessed on Ehrlich's ascites carcinoma (EAC) bearing mice model. Fluorescent microscopy and expression of PARP-1, Bax, and Bcl-2 through qPCR were performed to evaluate apoptosis. MLL and MLB extracts show promising antioxidant property with an IC50 value of 186.76 µg/ml and 352.97 µg/ml, respectively, with a decent LD50 value of 99.16 µg/ml and 92.54 µg/ml for MLL and MLB extract, respectively, indicated notable cytotoxicity. Cell growth inhibition was observed using MLL and MLB extracts were 68.33% and 48.66%, respectively. The morphological alteration, DNA fragmentation, and differential expression of Bax, Bcl-2, and PARP-1 confirm the induction of the intrinsic pathway of apoptosis. PRACTICAL APPLICATIONS: Plant-based medicine always plays a tremendous role in preventing several fatal diseases like cancer. The study evaluated the anticancer activity of a wild-type mulberry. Moreover, the potent antioxidant activity of the plant makes it possible to be a great candidate for cancer remedy. Besides, the molecular expression of the genes related to apoptosis confirms the plant's bioactive compounds could be a drug lead to neoplastic cells in the future. Presences of an immense antioxidant properties urge that they can be contribute in cancer treatment through the cell death pathways.

In-vivo antiproliferative activity of Morus latifolia leaf and bark extracts against Ehrlich's ascites carcinoma.

Cancer is the second death causing disease all over the world and until today 100 different types of cancer have been identified whose treatment methods consist of serious side effects on human body. To reduce the frequency of adverse effects of cancer treatment, nowadays plant derived natural components are getting priority. The plant Morus latifolia is widely available in northern part of Bangladesh. The earlier researches suggested that popular varieties of some Morus sp. like Morus alba, Morus indica etc. have good anti-proliferative activity. Hence, this study was designed to evaluate the anti-proliferative activity of leaf and bark extracts of M. latifolia against Ehrlich's ascites carcinoma (EAC) in vivo. The leaf and bark extracts of M. latifolia were used in several bioassays including Brine shrimp lethality test, hemagglutination activity test, antioxidant activity test, and cell growth inhibition test. Besides, fluorescence microscopy was performed to study apoptotic features in EAC cells, and molecular analysis like real-time PCR were also conducted. The results of Brine shrimp lethality test, hemagglutination activity test, and antioxidant activity assay supported the cell growth inhibition capability of leaf and bark extracts which was confirmed by in vivo cell growth inhibition bioassay. Moreover, the experimental extracts were able to induce cell apoptotis through altering the expression pattern of Bcl-2 and Bax genes. All of the results of this study suggest that several noble compounds are present in M. latifolia plant extracts which are capable for healing cancer cells.

BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis.

For tumors to progress efficiently, cancer cells must overcome barriers of oxidative stress. Although dietary antioxidant supplementation or activation of endogenous antioxidants by NRF2 reduces oxidative stress and promotes early lung tumor progression, little is known about its effect on lung cancer metastasis. Here, we show that long-term supplementation with the antioxidants N-acetylcysteine and vitamin E promotes KRAS-driven lung cancer metastasis. The antioxidants stimulate metastasis by reducing levels of free heme and stabilizing the transcription factor BACH1. BACH1 activates transcription of Hexokinase 2 and Gapdh and increases glucose uptake, glycolysis rates, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of mouse and human lung cancer cells. Targeting BACH1 normalized glycolysis and prevented antioxidant-induced metastasis, while increasing endogenous BACH1 expression stimulated glycolysis and promoted metastasis, also in the absence of antioxidants. We conclude that BACH1 stimulates glycolysis-dependent lung cancer metastasis and that BACH1 is activated under conditions of reduced oxidative stress.

In Vivo Anticancer Activity of Basella alba Leaf and Seed Extracts against Ehrlich's Ascites Carcinoma (EAC) Cell Line.

Cancer is a class of diseases characterized by uncontrolled cell growth. The current treatment options of cancer are radiotherapy, chemotherapy, hormone therapy, and surgery, where all of them have unpleasant side effects. Due to their adverse side effects, it is challenging to develop new drug for cancer treatment. Hence, the scientists are trying to seek for noble compounds from natural sources to treat cancer. Therefore, in the present investigation, a widely consumable vegetable Basella alba was subjected to evaluate its antiproliferative effect along with molecular signaling of apoptosis in Ehrlich ascites carcinoma (EAC) cell line. Cell growth inhibition was determined by haemocytometer whereas apoptosis of cancer cells were studied by florescence microscope using Hoechst-33342 stain and result was supported by DNA fragmentation and certain cancer related genes expression through PCR analysis. B. alba leaf and seed extract exhibit a considerable scavenging activity in comparison to a standard antioxidant BHT. Moreover, the leaf and seed extracts were able to agglutinate 2% RBC of goat blood at minimum 12.5µg/ml and 50.0µg/ml concentration, respectively. A significant cytotoxic activity was also found in both leaf and seed extract. In haemocytometic observation, the leaf and seed extracts exhibit about 62.54±2.41% and 53.96±2.34% cell growth inhibition, respectively, whereas standard anticancer drug Bleomycin showed 79.43±1.92% growth inhibition. Morphological alteration under fluorescence microscope showed nuclear condensation and fragmentation which is the sign of apoptosis. Apoptosis induction was also confirmed by DNA laddering in leaf and seed treated EAC cells. Upregulation of the tumor suppressor gene P53 and downregulation of antiapoptotic gene Bcl-2 enumerate apoptosis induction. Therefore, current study manifested that leaf and seed extracts of B. alba have antiproliferative activity against EAC cell line and can be a potent source of anticancer agents to treat cancer.