**1. Introduction**

In the last years, novel and effective immunotherapies for patients with different tumor types are becoming clinically important, because of the remarkable clinical efficacy observed with several immune checkpoint inhibitors such as cytotoxic T lymphocyte antigen 4 (CTLA-4) and the programed death receptor 1 (PD-1) or its ligand (PD-L1) [1–12]. Whereas anti-CTLA-4 antibodies (ipilimumab and tremelimumab), anti-PD-1 antibodies (nivolumab and pembrolizumab), and anti-PD-L1 antibodies (atezolizumab, avelumab, and durvalumab) have produced remarkable results, increasing the survival prognosis in many cancer types, it is still unknown why some tumors do not respond to or relapse after this type of treatment. In this way, increased observations suggest that tumors rich in tumor-associated immune cells (TAICs) may respond to therapies targeting immune system inhibitory or stimulatory mechanisms, and tumors with non-TAICs may require additional interventions aimed at promoting optimal inflammation and innate immune activation in the tumor microenvironment [13–15]. Indeed, characterization of different immune checkpoints as well as tumor microenvironment in patients with cancer has become

a fundamental step in providing evidence for the presence of distinct immunologic phenotypes, based on the presence or absence of various immune cells [1, 16, 17] that can predict the response to the therapy. In this way, the study of immune checkpoints and TAICs and their interaction prompt the need for multiplexed analyses of tumor tissues. To address this need, in the last years, multiplex imaging platforms have emerged as an important tool to provide critical information about cancer microenvironment, prognosis, therapy, and relapse [18–22]. Different components in the tumor microenvironment can be examined simultaneously using multiplex methodologies, providing insight into biological cross-talk present at the tumor-host interface and from subcellular levels to entered cell populations. In addition, the precision of these new techniques can be used to evaluate the special distribution of multiple biomarkers detected simultaneously, and their coexpressions or interactions between cells are becoming a essential tool to study tumor tissues [22] and to ultimately enhance disease diagnosis and better inform timely patient care [23].

Multiplex technologies are being used to identify the presence of multiple biological markers as immune checkpoint and TAICs on a single tissue sample [24]. The multiplex imagining techniques provide unique biological information that in many cases cannot be obtained by other imaging methods or by single immunohistochemistry (IHC) techniques. As mentioned, individual cells can be accessed with extraordinary fidelity equal to that achievable in the bulk population, such that even rare cell populations can be studied to understand their important role in translational research, and this knowledge can be applied in cancer prevention and treatment. In this chapter, we will discuss one of the most reliable and a very well-known methodology to identify simultaneous biomarkers in formalin-fixed, paraffin-embedded (FFPE) specimens as well as its imaging analysis platform as an important tool for potential application in future cancer immunotherapy biomarker discoveries.
