One of the primary roles of the immune system is the specific identification and elimination of tumor cells on the basis of their expression of tumor-specific antigens or molecules induced by cellular stress. This process is referred to as tumor immune surveillance. In this process the immune system recognizes malignant and/or pre-malignant cells and removes them. However, tumor cells do escape from tumor immune surveillance, and therefore, therapies targeted to enhance antitumor immunity is currently in development.

Blockade of immune checkpoints  is the most promising approach to activate therapeutic antitumour immunity. Immune checkpoints refer to a group of inhibitory pathways connected into the immune system that are important for maintaining self-tolerance. In peripheral tissues immune surveillance also modulates the duration and amplitude of physiological immune responses in order to minimize collateral tissue damage. Studies have suggested that tumor cells adopt many immune-checkpoint pathways as a major mechanism of immune resistance. Immune checkpoint receptors cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, also known as CD152) and programmed death 1 (PD-1) receptor appear to play important roles in antitumor immunity and have been most actively studied in the context of clinical cancer immunotherapy.

monoclonal3CTLA-4 is expressed on T cells and down modulates the amplitude of T cell activation. Several preclinical studies demonstrated significant antitumor responses following blockade of CTL4-A with limited immune toxicities. This led to the development of two fully humanized  CTLA-4 antibodies ipilimumab and tremelimumab. In clinical trials, ipilimumab demonstrated survival benefits for patients with metastatic melanoma, and was approved by the US Food and Drug Administration (FDA) for the treatment of advanced melanoma in 2010.

On the other hand, PD-1limits T cell effector functions within tissues. Tumor  cells block antitumor immune responses in the tumor microenvironment by upregulating ligands (PDL1 and PDL2) for PD1. Several studies detected increased PD1 expression by tumor infiltrating lymphocytes and the increased expression of PD1 ligands in melanoma, ovarian, lung, renal-cell cancers and in lymphomas. This provided an important rationale to target PD1 in order to enhance antitumor immunity. The fully human antibody nivolumab was found to produce durable objective responses in patients with melanoma, renal-cell cancer, and non-small-cell lung cancer.

Even though individual blocking of CTLA-4 and PD-1 have shown substantial clinical antitumor activity, studies suggest that blocking a single inhibitory receptor only leads to up-regulation of the unblocked pathway. Therefore, in order  to enhance antitumor immunity within the tumor microenvironment it appears to require simultaneous blockade of multiple negative co-stimulatory receptors. In preclinical studies, concurrent inhibition of CTLA-4 and PD-1 resulted in more pronounced antitumor activity than blockade of either pathway alone. On the basis of these observations, a phase I study was conducted to investigate the safety and efficacy of combined inhibition of CTLA-4 and PD-1in advanced melanoma patients and published recently in The New England Journal of Medicine (July 11, 2013). In their study, Wolchok and collagues (2013) treated 53 patients concurrently, and 33 patients sequentially with nivolumab and ipilimumab. Rapid responses were observed in concurrent-regimen cohorts as compared with sequential-regimen cohorts. The objective response rate in the concurrent-regimen cohorts was 40% along with 53% patients exhibited tumor regression of 80% or more. The objective response rate in the sequenced-regimen cohorts was 20% and 13% patients had tumor regression of 80% or more. In both groups, treatment related adverse events were managed with the use of immunosuppressants.

Collectively this study suggested that combined blockade of CTLA-4 and PD-1 would be more effective to enhance antitumor immunity compared to single inhibition of either CTLA-4 or PD-1.


1.  Swann, J.B. and M.J. Smyth, Immune surveillance of tumors. J Clin Invest, 2007. 117(5): p. 1137-46.

2.   Pardoll, D.M., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012. 12(4): p. 252-64.

3.   Topalian, S.L., et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med, 2012. 366(26): p. 2443-54.

4.   Wolchok, J.D., et al., Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med, 2013. 369(2): p. 122-33.

Techniques in Cytology – Cytospin: Cytospin Cell Resuspension Solution

In this blog series opening article, I discussed how cytospins could be used to separate cancer cells from non-cancer cells1. This cytology method and subsequent staining of the resultant cells are a key component of diagnosis and screening of diseases such as cancer. The cytospin technique can be used on any single cell suspensions of any source such as peripheral blood mononuclear cells (PBMCs), effusions, cerebral spinal fluid (CSF), bronchial lavages, fine-needle aspirates, culture cells, etc.

In cytospins, single cell suspensions are spun onto a microscope slide by use of a cytocentrifuge. A cytocentrifuge spins cells at an angle, at low speeds, and accelerates and decelerates gradually. The fluid from the suspension is absorbed onto filter paper while the centrifuge is spinning. This allows the cells to adhere to the slide in a monolayer format. The cell settling rate is determined by the rotational speed of the centrifuge and the size and density of the cells.  Large or dense numbers of cells settle quickly. Small or sparse numbers of cells settle slowly.

One question often asked is what is the optimum resuspension solution for cells that will undergo the cytospin process? The type of resuspension solution used is dependent on the cells.  A good rule of thumb is to use whatever solution will keep the cells alive and healthy throughout the cytospin procedure. The best morphology and subsequent staining of the cytospin cells are generated from cells that are freshly harvested. However, as soon as cells are removed from the body, they begin to die. In order to delay the cell death and protect the cells during the cytospin process, cells are resuspended in tissue culture media or phosphate buffered saline (PBS) with 1% – 10% bovine serum albumin (BSA) and/or 1% – 2% serum such as fetal calf serum (FCS). The tissue culture media and PBS provide a pH balanced liquid and ion replacement for the cell’s native environment. The BSA and serum provide a source of protein as a nutrient to keep the cells healthy and alive for a relatively short amount of time. BSA and serum also stabilizes enzymes to delay internal protein and nucleic acid degradation, which leads to a cascade of events that eventually destroys the cell.

So, should you use tissue culture media or PBS boosted with protein? Again, this depends on the cells and the answer to this question must be determined empirically. If the cells are coming directly from in vitro cell culture then the answer is easy. Keep the cells in the cell culture media that they were culture in. Unless you are interested in seeing on your cytospin slide everything that was in the cell culture well, it is suggested that you wash your cells in fresh media to remove any debris or dead cells before you cytospin. If the cells will be taken directly from the host/patient and then processed for cytospin, determine how long the cells will be out of the host/patient before they are actually taken through the cytospin process. If the cells will be taken through multiple washes to remove debris, other cells, or tissue structural components that are not of interest, it is best to keep the cells resuspended in tissue culture media. A good starting tissue culture media is Dulbecco’s Modified Eagle Medium (DMEM) with 10% FCS. If the cells will be processed immediately or only go through one wash, the cells could be resuspended in PBS with 1% BSA plus 1% FCS as a good starting solution.

My next post will continue with this series on the cytospin process as well as presenting tips for troubleshooting.

1              Ikeda, K., Tate, G., Suzuki, T., Kitamura, T. & Mitsuya, T. Diagnostic usefulness of EMA, IMP3, and GLUT-1 for the immunocytochemical distinction of malignant cells from reactive mesothelial cells in effusion cytology using cytospin preparations. Diagn Cytopathol 39, 395-401, doi:10.1002/dc.21398 (2011).