Background: Traditional immunohistochemistry (IHC) with chromogen detection has limited multiplex capacity, detecting at most 4 protein markers per tissue section simultaneously, thereby restricting comprehensive spatial analysis of valuable human biopsies. We developed and validated a robust serial IHC (sIHC) staining method to detect multiple antigens on a single kidney biopsy slide, maximizing data yield for diagnosing and studying complex kidney diseases. Methods: Formalin-fixed, paraffin-embedded kidney biopsy sections were subjected to repeated IHC/imaging cycles with antibody removal using an optimized sodium dodecyl sulfate-glycerol buffer stripping protocol. Images were then co-registered, and analysis was performed using a variety of methodologies, including color deconvolution, cell segmentation, and spatial clustering. Results: This optimized sIHC method successfully detected up to 20 antigens on a single slide. Combining image analysis and artificial intelligence software, for example with HALO (Indica Labs), the assay assembles high-dimensional images and enables quantitative histology and single-cell spatial analysis. Using this advanced method, we were able to identify rare cell populations, such as double-negative T cells, that are challenging to detect conventionally. Conclusion: We have developed a validated, high-capacity sIHC protocol that uses standard IHC procedures with commercially available, clinically validated off-the-shelf antibodies. This method is a valuable, cost-effective tool for obtaining extensive, high-dimensional single-cell-resolved spatial data from limited pathology samples, such as a human kidney biopsy.

Cancer and atherosclerotic cardiovascular disease (ASCVD) represent two of the leading causes of death worldwide. Increasingly, these two are being recognized as biologically related conditions rather than entirely segregated disease states. In addition to traditional risk factors such as aging, smoking, and obesity, chronic inflammation may be a key factor connecting the two illnesses. Endothelial dysfunction, oxidative stress, plaque progression, and thrombosis are all facilitated by inflammatory signaling in ASCVD. Similar pathways are known to contribute to cancer growth and invasion. Emerging epidemiologic data demonstrate increased cancer incidence among patients with cardiovascular disease, while cancer survivors and recipients of cardiotoxic therapies exhibit accelerated vascular disease. This narrative review aims to describe the bidirectional relationship between ASCVD and cancer. Targeting shared pathways using statins, colchicine, canakinumab, IL-6 inhibition, and lifestyle modification may provide dual benefits. Future biomarker-guided trials with integrated cardiovascular and oncologic endpoints are needed to clarify causality and optimize prevention and management.