Sound the alarm! How hemocyanins trigger immunity for cancer vaccines


The immune system is, in many cases, a well-oiled war machine. Its combination of a quick, general innate response and a specific adaptive response leading to memory protects us well from foreign enemies, like bacteria and viruses. But in the case of cancer, it’s the body’s own cells that are the enemy. How can the immune system “sound the alarm” to attack cells that don’t bear any of the classic signs of an invader? Cancer vaccines may be the answer. Research into these vaccines involves some of the same principles that have been successful in training the immune system to recognize microbes – show immune cells an antigen in combination with an immunostimulant, triggering a robust response to tumor antigens that would otherwise be perceived as harmless.

Scientists have long explored the use of hemocyanin from the keyhole limpet sea snail (KLH) as an immunogenic carrier protein in cancer vaccine research. Hemocyanin is a large metalloprotein in invertebrates that is responsible for carrying oxygen, analogous to hemoglobin in mammalian cells, although hemocyanin is not bound to cells. KLH has been shown to boost immune cells’ responses to tumor lysate or peptide (1) and is in use in a number of cancer vaccine clinical trials (2–3). Hemocyanins from other sea snails are also of interest, such as CCH, from Concholepas concholepas (4), and FLH from Fissurella latimarginata (5). To understand these molecules’ suitability in vaccine research, it’s important to dissect the varying responses they elicit from the immune system.

So what’s the best place to start when evaluating the differences in immune responses? Gene expression analysis is one powerful avenue for understanding immunity. By tracing the levels of the right messenger RNAs, we can get clues about the activation or suppression of immune signaling pathways, the involvement of particular pattern recognition receptors, or the activation of the Th1 or Th2 branches of the adaptive immune system. RNA-sequencing is on the rise, and provides terrific opportunities for de novo discovery and specificity to labs with a sequencer or core facility, while qPCR offers excellent dynamic range (the capacity to detect both very low-expressing and very high-expressing genes in one experiment) and accessibility.

In a recent study, Zhong et al. used gene expression profiling via qPCR array, in conjunction with ELISA and other techniques, to help define the types of responses triggered in mouse peritoneal macrophages by KLH, CCH and FLH (6). Macrophages are major players in immune responses, phagocytosing microbes and debris, secreting proinflammatory or anti-inflammatory molecules like cytokines and chemokines, and presenting antigens to T cells. Their in vitro response to each hemocyanin is a useful piece of information for predicting what the immune environment triggered by these molecules would look like in the body. The team incubated the cells with each type of hemocyanin at various timepoints, and then isolated total RNA with RNeasy and assessed mRNA levels of cytokines and chemokines with an RT² Profiler PCR Array.

The hemocyanins elicited distinct gene expression profiles after phagocytosis by macrophages. Each led to a downregulation in transcripts for cytokines IL4 and IL5, and they also all induced upregulation of CD40L, which the authors speculated might be related to their nonspecific antitumor activity. However, FLH-exposed macrophages produced more proinflammatory cytokines/chemokines, such as CCL3 and IFN-gamma, compared to KLH and especially compared to CCH, suggesting a stronger ability to provoke inflammatory responses. FLH alone also downregulated TGF-beta2 transcript. Protein expression analysis showed consistency with the mRNA analysis for some cytokines, but other cytokines that were shown as expressed at the mRNA level didn’t exhibit protein expression, such as IL-6 and IL-12 p40, possibly due to posttranscriptional regulation.

So what does all this mean for cancer vaccine research? In the end, these 3 hemocyanins all trigger different magnitudes of immune-related gene expression response from macrophages. Different signaling pathways appear to be triggered by each, possibly indicating that they use different receptors on the macrophage surface. The authors note that FLH’s stronger proinflammatory induction capabilities may explain why it has antitumor activity without previous priming, in contrast to the other two hemocyanins. These differences will need to be taken into account when deciding what carrier protein to use in future vaccine development. As is often the case in biomedical research, questions beget questions, and more work is needed to fully understand how these proteins work. But given their promise in cancer vaccine research thus far, we can be confident that more information will be forthcoming.

How can you investigate immunity using qPCR? QIAGEN’s qPCR workflow can take you all the way from sample to insight with RNeasy/miRNeasy for RNA isolation, reverse transcription and preamplification solutions, and qPCR arrays that range from the pathway-specific (inflammatory cytokines, lymphocyte activation, DC/APC responses and more) to the fully customizable.

Want to know more about qPCR technology? Download the webinar slides on Slideshare!



  1. 1. Shimizu, K. et al. (2001) Enhancement of tumor lysate- and peptide-pulsed dendritic cell-based vaccines by the addition of foreign helper protein. Cancer Res. 61, 2618. Link
  2. 2., NCT02543749 
  3. 3., NCT01426828
  4. 4. Moltedo, B. et al. (2006) Immunotherapeutic effect of Concholepas hemocyanin in the murine bladder cancer model: evidence for conserved antitumor properties among hemocyanins. J. Urol. 176, 2690. Link
  5. 5. Arancibia, S. et al. (2014) A novel immunomodulatory hemocyanin from the limpet Fissurella latimarginata promotes potent anti-tumor activity in melanoma. PLoS One 9, e87240. Link
  6. 6. Zhong, T.Y. et al. (2016) Hemocyanins stimulate innate immunity by inducing different temporal patterns of proinflammatory cytokine expression in macrophages. J. Immunol. 196, 4650. Link
Ali Bierly

Ali Bierly, PhD is a Global Market Manager in Translational Sciences at QIAGEN, and has written on a number of scientific topics in the biotech industry as the author of QIAGEN's Reviews Online. She received her PhD from Cornell University in 2009, studying the immune response to a protozoan parasite, Toxoplasma gondii. Ali has a keen interest in the emerging importance of microRNA and other circulating nucleic acids as biomarkers for disease.

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