**3. Image approaches and data analysis**

*Immunohistochemistry - The Ageless Biotechnology*

staining in control tissues. The use of specific, very well-standardized, and validated antibodies, as well as the careful use of other components, as right antibody titration, incubation time, and antigen retrieval during staining, is important to obtain good and reproducible results using different panels [27]. The use of very well-known control tissues during each staining and for each created panel that allows all the markers is important and essential to detect possible staining errors during the process in each mIF panel. Properties of the FFPE material, such as sample age, method of preservation, storage conditions, and tissue type, are very important factors to be considered to obtain high-quality mIF staining and good data. Pathologists play a key role in making sure that tissue samples collected are appropriate for diagnostic and research purposes. The tissues need to be processed adequately, that is, fixed in 10% formalin and stored in good conditions to avoid antigenic deterioration that can influence the process when targeting several proteins using this methodology. Type of tissue, is another important factor to be considered, sometimes as a limitation factor for a quality staining with this technique. We observed that some tissues that have abundant fat as breast tissue or cartilage in some type of cancers or bone component that were submitted a decalcification procedures, are more challenges during the mIF staining, showing frequently artifacts of staining like background, folds, caused by tissue detached and unspecific or not clear staining on the cells, causes by the decalcification procedures. Antibodies with very good performance in decalcified tissues are limited, and those need an exhaustive validation in IHC before creating a new panel to stain these samples. No less important, the size of the sample is another factor to be considered during mIF staining; small biopsies as core needle biopsies (CNBs) less than 1.0 × 0.2 cm are more challenging and have high probability to be lost during standard mIF staining than bigger tissues as whole sections (~1.0 × 1.0 cm). The minimum number of malignant tumor cells required for mIF marker analysis has not been well established and is another factor to be considered during staining and analysis.

**58**

**Table 1.**

**Specimen type**

Whole section

Whole section

Small biopsies

Small biopsies

Small biopsies

TMA (by core)

**Size (cm) Viable** 

**tumor cells (N)** **Necrosis (%)**

>1.0 × 1.0 >100 0 or >10 >50 of any component 80% in our

<1.0 × 0.2 >100 <10 <50 of any component 70% in our

<1.0× 0.2 <100 >10 >50 of any component 50% in our

>1.0 × 1.0 >100 0 or <10 0 or <10 of any

>1.0 × 0.2 >100 0 or <10 0 or <5 of any

>0.1 >100 0 or <5 0 or <5 of any

*A preliminary quality control to establish the samples by a pathologist is strongly recommended to optimize the preparation of tissue for multiplex immunofluorescence staining and ultimately to guaranty a quality data. Each case needs to be considered separately and can be influenced by several characteristics. In general, the quality of the samples including the fixation process of the FFPE tissues, storage and cutting procedures, will influence the quality of multiplex staining (\*). There are, however, according to our experience, different tissue characteristics that need to be considered as challenges for staining and analysis, and these are considered sometimes as limitations of the staining. By understanding much better these tissue limitations, we can avoid wasted effort, resources, and funds of* 

*the laboratory as well as preserve the high-quality data obtained by this technique.*

*Quality criteria's samples for multiplex immunofluorescence.*

**Fat/cartilage/bone (%) Adequacy** 

component

component

component

**for mIF staining\***

100% in our series

series

100% in our series

series

series

100% in our series

Although the methodology of TSA is available for FFPE material and can enable multiparametric readouts from a single tissue section, they sometimes have limited scalability and throughput, related to limited number of markers allowed per panel compared with other multiplex methodologies like imaging mass cytometry and multiplexed ion beam imaging [35, 36]. The scanner system (**Figure 2**) Vectra® [27] from PerkinElmer provides high quality of scanning with high-resolution and multiband filter cubes that provide greater flexibility associated with the multispectral camera, to match with the sample. The new generation of scanner Polaris™ (PerkinElmer) scan system supports multiple filters using tunable LED excitation, similar to confocal microscope, and the captured signals are assembled in a composite image [37]. After acquiring the panoramic low-magnification images at ×4 or ×10, the specimens can be sampled using different ROI sizes by the phenochart (PerkinElmer) software viewer to scan high-resolution images at ×20 or ×40. Although, the scanner system Vectra®-Polaris™ can capture different regions of interest (ROIs) using the filters and the multispectral camera at high quality resolution [36], it is still impossible to accelerate the process of scanning or scan the whole tissue section as a unique image for the analysis. The time for scanning the sample is variable and depends on the number of markers used in the panel, number of ROIs captured per sample, and size of the ROI and can take from minutes to several hours according these parameters [38] (**Table 2**). According our experience, the TSA


*The time for scanning the sample is variable and depends on the number of markers used in the panel, number of regions of interest (ROI) captured per sample, and size of the ROI using Vectra® or PolarisTM system, as well as whether the system stores the image locally or in a server.\* Available only in Vectra®.*

#### **Table 2.**

*Approximate time for image scanning using Vectra® or the PolarisTM scanner system.*


#### **Table 3.**

*Image analysis software systems available for multiplex immunofluorescence.*

staining system for mIF when combined with multispectral image analysis software, such as InForm (PerkinElmer), can provide a powerful tool for analysis of multiple markers in one single slide [21, 39]. However, there are many available software in the market that can be used for the analysis of mIF images generated by the InForm software from the Vectra®-Polaris™ scanner systems, and it is important to know that the InForm software is essential to generate the individual unmixed tyramide fluorochrome with a positive signal without noise or aberrant background staining and with high resolution performance across the different ROIs from the scanning systems [40]. For the analysis, image analysis software need to be accessible (**Table 3**), with easy automated capabilities of detection, including tissue segmentation, compartmentalization of the staining (e.g., nuclear, membranous, or cytoplasmic) (**Figure 2**), and spatial colocalization of cell distribution, critically important to study different markers included in different panels (**Figure 3**). In the same way, comprehensive evaluation using this different image analysis software is needed not only for clear antigen demarcation and good staining procedures but also for good interpretation of the results. Pathologists are very important and need to standardize the possible interobserved variation [41, 42] when using different image analysis platforms during the colocalization of proteins.

### **4. Multiplex immunofluorescence staining from translational research**

Despite the evolution in the last years, in different levels of cancer research, concerning prevention, diagnosis, therapeutic options, and follow-up methods, cancer diseases are still the major public health problem worldwide [43]. Profiling immune cells is currently a powerful metric for tumor subclassification and predicting clinical outcomes. A great variety of cancer research screening tools is applied to diagnose tumors and has been established for different tumors. Simultaneous quantification of more than one biomarker at the same time has become more and more interesting in cancer research using different multiplex technologies. Multiplex TSA can allow different biomarkers in one single slide, targeting different

**61**

**Figure 3.**

*(D) lung squamous cell carcinoma. ×20 magnification.*

*Immune Cell Profiling in Cancer Using Multiplex Immunofluorescence and Digital Analysis…*

systemic processes, such as inflammation, immunocheckpoints, angiogenesis, or cell death using tumor markers (**Figure 3**), to improve cancer prevention, diagnostic accuracy, and treatment. We demonstrated that this method can offer important advantages, such as high-throughput performance, low material requirement, wide range of applications, and cost- and time-effective multiplex for several parameters in different panels [23, 44, 45]. Several biomarkers can be cancer-specific since malignant cells of different histologic types can produce different patterns of

*Microphotographs of representative examples of multiplex immunofluorescence in tumor tissues using different markers, (A) lung adenocarcinoma, (B) lung squamous cell carcinoma, (C) malignant melanoma, and* 

*DOI: http://dx.doi.org/10.5772/intechopen.80380*

*Immune Cell Profiling in Cancer Using Multiplex Immunofluorescence and Digital Analysis… DOI: http://dx.doi.org/10.5772/intechopen.80380*

**Figure 3.**

*Immunohistochemistry - The Ageless Biotechnology*

Tissuemorph

Visiopharm Visimoph

**Table 3.**

platforms during the colocalization of proteins.

staining system for mIF when combined with multispectral image analysis software, such as InForm (PerkinElmer), can provide a powerful tool for analysis of multiple markers in one single slide [21, 39]. However, there are many available software in the market that can be used for the analysis of mIF images generated by the InForm software from the Vectra®-Polaris™ scanner systems, and it is important to know that the InForm software is essential to generate the individual unmixed tyramide fluorochrome with a positive signal without noise or aberrant background staining and with high resolution performance across the different ROIs from the scanning systems [40]. For the analysis, image analysis software need to be accessible (**Table 3**), with easy automated capabilities of detection, including tissue segmentation, compartmentalization of the staining (e.g., nuclear, membranous, or cytoplasmic) (**Figure 2**), and spatial colocalization of cell distribution, critically important to study different markers included in different panels (**Figure 3**). In the same way, comprehensive evaluation using this different image analysis software is needed not only for clear antigen demarcation and good staining procedures but also for good interpretation of the results. Pathologists are very important and need to standardize the possible interobserved variation [41, 42] when using different image analysis

**Vendor Program name Method Availability**

measurement, and statistical analysis

Signal intensity, area, counting objects,

contouring, measuring associated signals

proximity, spatial analysis

statistical analysis

Spot Imagine Spot advanced Color-based colocalization Licensed

NIH Image J Color-based, user interactive segmentation Free HistoRx AQUAnalysis Signal intensity per unit area and per layer Licensed

Licensed

Licensed

Licensed

Licensed

Free

Licensed

segmentation

PerkinElmer InForm Color-based colocalization, tissue, cell

Definiens Tissue Studio Imaging segmentation, marker intensity

Indica Labs HALO Membrane, colocalization, immune cell

FARSIGHT Nucleus Editor Multichannel-based object identification/ toolkit

CompuCyte iCyte Nucleus segmentation or phantom

*Image analysis software systems available for multiplex immunofluorescence.*

**4. Multiplex immunofluorescence staining from translational research**

Despite the evolution in the last years, in different levels of cancer research, concerning prevention, diagnosis, therapeutic options, and follow-up methods, cancer diseases are still the major public health problem worldwide [43]. Profiling immune cells is currently a powerful metric for tumor subclassification and predicting clinical outcomes. A great variety of cancer research screening tools is applied to diagnose tumors and has been established for different tumors. Simultaneous quantification of more than one biomarker at the same time has become more and more interesting in cancer research using different multiplex technologies. Multiplex TSA can allow different biomarkers in one single slide, targeting different

**60**

*Microphotographs of representative examples of multiplex immunofluorescence in tumor tissues using different markers, (A) lung adenocarcinoma, (B) lung squamous cell carcinoma, (C) malignant melanoma, and (D) lung squamous cell carcinoma. ×20 magnification.*

systemic processes, such as inflammation, immunocheckpoints, angiogenesis, or cell death using tumor markers (**Figure 3**), to improve cancer prevention, diagnostic accuracy, and treatment. We demonstrated that this method can offer important advantages, such as high-throughput performance, low material requirement, wide range of applications, and cost- and time-effective multiplex for several parameters in different panels [23, 44, 45]. Several biomarkers can be cancer-specific since malignant cells of different histologic types can produce different patterns of

#### **Figure 4.**

*Microphotographs of representative examples of spatial-distribution visualization of different phenotypes analyzed. (A) distribution of individual cells using X and Y positions, (B) spatial localization of selected cells, and (C to F) distance measurements between malignant cells (MCs) and different cell populations.*

proangiogenic factors, growth factors, and immune cells that are tumor related. The study of biomarker panels (**Figure 3**) and its spatial distribution (**Figure 4**) can be used for early diagnosis and assessment of therapy response [46]. This methodology can represent an ideal method to realize personalized therapies using efficient mIF panels and help to understand much better the cancer microenvironment,

**63**

provided the original work is properly cited.

Anderson Cancer Center, Houston, Texas, USA

\*Address all correspondence to: erparra@mdanderson.org

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Translational Molecular Pathology, The University of Texas MD

*Immune Cell Profiling in Cancer Using Multiplex Immunofluorescence and Digital Analysis…*

highlighting the benefit for exploring immune evasion mechanisms and finding potential biomarkers that allow researchers to assess the mechanism of action and

The detection of multiple markers in the same tissue section can provide important and efficient means to apply this technology in disease diagnosis, prevention, and translational research. Multiplex immunoflourescence platforms have emerged more and more from translational research labs toward the clinic, increasing the opportunity to study and understand much better the tumor-immune interactions. This methodology and different image analysis strategies can give important information about immune cells' coexpression and their spatial-pattern distribution in the tumor microenvironment. Development of multiplex immunoflourescence based-TSA system requires a very well-trained multidisciplinary team including pathologists, oncologists, and immunologists. In addition, this methodology requires automation to provide efficient and fast information as well as easy analysis

The author would like to acknowledge the people that work in the laboratory of multiplex immunofluorescence, Mei Jang, Tong Li, Aerole Tanhemon, and Barbara Mino; the pathology team that work in the multiplex image analysis; as well as the chair of the Department of Translational Molecular Pathology, Dr. Ignacio Wistuba.

methodologies for research pathologists that currently use this method.

The author does not have any type of competing interest.

predict and track response [47].

*DOI: http://dx.doi.org/10.5772/intechopen.80380*

**5. Conclusion**

**Acknowledgements**

**Conflict of interest**

**Author details**

Edwin Roger Parra

*Immune Cell Profiling in Cancer Using Multiplex Immunofluorescence and Digital Analysis… DOI: http://dx.doi.org/10.5772/intechopen.80380*

highlighting the benefit for exploring immune evasion mechanisms and finding potential biomarkers that allow researchers to assess the mechanism of action and predict and track response [47].
